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Scholte, Florine E. M., Ali Tas, Irina C. Albulescu, Eva Žusinaite, Andres Merits, Eric J. Snijder, and Martijn J. van Hemert. "Stress Granule Components G3BP1 and G3BP2 Play a Proviral Role Early in Chikungunya Virus Replication." Journal of Virology 89, no. 8 (February 4, 2015): 4457–69. http://dx.doi.org/10.1128/jvi.03612-14.
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ABSTRACTStress granules (SGs) are protein-mRNA aggregates that are formed in response to environmental stresses, resulting in translational inhibition. SGs are generally believed to play an antiviral role and are manipulated by many viruses, including various alphaviruses. GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1) is a key component and commonly used marker of SGs. Its homolog G3BP2 is a less extensively studied SG component. Here, we demonstrate that Chikungunya virus (CHIKV) infection induces cytoplasmic G3BP1- and G3BP2-containing granules that differ from bona fide SGs in terms of morphology, composition, and behavior. For several Old World alphaviruses it has been shown that nonstructural protein 3 (nsP3) interacts with G3BPs, presumably to inhibit SG formation, and we have confirmed this interaction in CHIKV-infected cells. Surprisingly, CHIKV also relied on G3BPs for efficient replication, as simultaneous depletion of G3BP1 and G3BP2 reduced viral RNA levels, CHIKV protein expression, and viral progeny titers. The G3BPs colocalized with CHIKV nsP2 and nsP3 in cytoplasmic foci, but no colocalization with nsP1, nsP4, or dsRNA was observed. Furthermore, G3BPs could not be detected in a cellular fraction enriched for CHIKV replication/transcription complexes, suggesting that they are not directly involved in CHIKV RNA synthesis. Depletion of G3BPs did not affect viral entry, translation of incoming genomes, or nonstructural polyprotein processing but resulted in severely reduced levels of negative-stranded (and consequently also positive-stranded) RNA. This suggests a role for the G3BPs in the switch from translation to genome amplification, although the exact mechanism by which they act remains to be explored.IMPORTANCEChikungunya virus (CHIKV) causes a severe polyarthritis that has affected millions of people since its reemergence in 2004. The lack of approved vaccines or therapeutic options and the ongoing explosive outbreak in the Caribbean underline the importance of better understanding CHIKV replication. Stress granules (SGs) are cytoplasmic protein-mRNA aggregates formed in response to various stresses, including viral infection. The RNA-binding proteins G3BP1 and G3BP2 are essential SG components. SG formation and the resulting translational inhibition are generally considered an antiviral response, and many viruses manipulate or block this process. Late in infection, we and others have observed CHIKV nonstructural protein 3 in cytoplasmic G3BP1- and G3BP2-containing granules. These virally induced foci differed from true SGs and did not appear to represent replication complexes. Surprisingly, we found that G3BP1 and G3BP2 were also needed for efficient CHIKV replication, likely by facilitating the switch from translation to genome amplification early in infection.
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Yang, Ziwei. "Expression of human G3BP1 in E. coli." E3S Web of Conferences 292 (2021): 03087. http://dx.doi.org/10.1051/e3sconf/202129203087.
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G3BP1 (Ras-GTPase-activating protein SH3 domain-binding protein) is responsible for normal RNA stress granule (SG) assembly and overexpressed in many cancer cells. Deletion of G3BP1 decreases the number and size of SGs. SGs are complex of RNA and proteins that stall translation of protein in response to stress. Given the function of G3BP1 in stress granule assembly and tumor suppression, it is believed that G3BP1 regulates cell growth and proliferation as well. Here, I constructed the recombinant protein expression vector and systemically optimized condition for the expression of human G3BP1 protein in E. coli. This research should be useful for investigating further functional analysis and atomic structure of G3BP1.
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Hinton, Shantá D., Michael P. Myers, Vincent R. Roggero, Lizabeth A. Allison, and Nicholas K. Tonks. "The pseudophosphatase MK-STYX interacts with G3BP and decreases stress granule formation." Biochemical Journal 427, no. 3 (April 14, 2010): 349–57. http://dx.doi.org/10.1042/bj20091383.
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MK-STYX [MAPK (mitogen-activated protein kinase) phospho-serine/threonine/tyrosine-binding protein] is a pseudophosphatase member of the dual-specificity phosphatase subfamily of the PTPs (protein tyrosine phosphatases). MK-STYX is catalytically inactive due to the absence of two amino acids from the signature motif that are essential for phosphatase activity. The nucleophilic cysteine residue and the adjacent histidine residue, which are conserved in all active dual-specificity phosphatases, are replaced by serine and phenylalanine residues respectively in MK-STYX. Mutations to introduce histidine and cysteine residues into the active site of MK-STYX generated an active phosphatase. Using MS, we identified G3BP1 [Ras-GAP (GTPase-activating protein) SH3 (Src homology 3) domain-binding protein-1], a regulator of Ras signalling, as a binding partner of MK-STYX. We observed that G3BP1 bound to native MK-STYX; however, binding to the mutant catalytically active form of MK-STYX was dramatically reduced. G3BP1 is also an RNA-binding protein with endoribonuclease activity that is recruited to ‘stress granules’ after stress stimuli. Stress granules are large subcellular structures that serve as sites of mRNA sorting, in which untranslated mRNAs accumulate. We have shown that expression of MK-STYX inhibited stress granule formation induced either by aresenite or expression of G3BP itself; however, the catalytically active mutant MK-STYX was impaired in its ability to inhibit G3BP-induced stress granule assembly. These results reveal a novel facet of the function of a member of the PTP family, illustrating a role for MK-STYX in regulating the ability of G3BP1 to integrate changes in growth-factor stimulation and environmental stress with the regulation of protein synthesis.
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Somasekharan, Syam Prakash, Amal El-Naggar, Gabriel Leprivier, Hongwei Cheng, Shamil Hajee, Thomas G. P. Grunewald, Fan Zhang, et al. "YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1." Journal of Cell Biology 208, no. 7 (March 23, 2015): 913–29. http://dx.doi.org/10.1083/jcb.201411047.
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Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5′ untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.
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Somasekharan, Syam Prakash, Fan Zhang, Neetu Saxena, Jia Ni Huang, I.-Chih Kuo, Caitlin Low, Robert Bell, et al. "G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress." Nucleic Acids Research 48, no. 12 (May 14, 2020): 6855–73. http://dx.doi.org/10.1093/nar/gkaa376.
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Abstract Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into RNA granules reduces their translation, but it remains unclear whether RBPs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress adaptation in cancer. To study transcript partitioning under cell stress, we catalogued mRNAs enriched in prostate carcinoma PC-3 cell PSs, as defined by polysome fractionation and RNA sequencing (RNAseq), and compared them to mRNAs complexed with the known SG-nucleator protein, G3BP1, as defined by spatially-restricted enzymatic tagging and RNAseq. By comparing these compartments before and after short-term arsenite-induced oxidative stress, we identified three major categories of transcripts, namely those that were G3BP1-associated and PS-depleted, G3BP1-dissociated and PS-enriched, and G3BP1-associated but also PS-enriched. Oxidative stress profoundly altered the partitioning of transcripts between these compartments. Under arsenite stress, G3BP1-associated and PS-depleted transcripts correlated with reduced expression of encoded mitochondrial proteins, PS-enriched transcripts that disassociated from G3BP1 encoded cell cycle and cytoprotective proteins whose expression increased, while transcripts that were both G3BP1-associated and PS-enriched encoded proteins involved in diverse stress response pathways. Therefore, G3BP1 guides transcript partitioning to reprogram mRNA translation and support stress adaptation.
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Hosono, Naoko, Mahfouz Reda, Bartlomiej P. Przychodzen, Chantana Polprasert, Latifa Zekri, Michael J. Clemente, Jamal Tazi, et al. "Haploinsufficiency and Deletions of G3BP1 on Chromosome 5q Result in Induction of TP53." Blood 124, no. 21 (December 6, 2014): 784. http://dx.doi.org/10.1182/blood.v124.21.784.784.
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Abstract Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common chromosomal abnormality in MDS. The extent of individual defects vary, which may account for observed clinical diversity. Del(5q) pathogenesis has been related to haploinsufficiency of genes contained in the common deleted regions (CDR), including RPS14, miR-145/146a and SPARC. Driver mutations or pathogenic microdeletions were not identified for these genes, suggesting that multiple genes must function in combination to promote clonal evolution and phenotypic heterogeneity. Hence, we performed a comprehensive analysis of somatic mutations in genes located on chromosome 5 (chr5), both in patients with diploid 5q and in those with del(5q), to clarify the role of germline and somatic mutations in disease pathogenesis. In parallel, expression analysis was performed to correlate haploinsufficiency with the frequency of mutational events, in particular for diploid 5q cases. Applying SNP-array karyotyping to samples from 146 patients with del(5q), the lesion was identified in 5q31.1q33.1. Two retained regions (CRRs) were also observed in q11.1q14.2 (CRR1) and q34qter (CRR2). Lower-risk MDS is frequently affected by CDR, while in higher-risk MDS and secondary AML CRR1/2 are commonly co-involved. Using whole exome sequencing, we identified 11 hemizygous mutations located within the deleted area in del(5q) (N=59), while in cases diploid for 5q (N=330), 243 heterozygous mutations were found. One of the mutations discovered on chr5q afflicted a gene G3BP1 (5q33.1), located within the CDR and present in 2 patients. Both were missense mutations (one heterozygous and the other homo/hemizygous). A mutant case showed good responses to lenalidomide even though diploid 5. In addition, other somatic mutations of driver genes including TET2, CUX1 and EZH2 were concomitantly observed. Whole transcriptome sequencing demonstrated hemizygous loss of G3BP1 resulting in haploinsufficiency. G3BP1 was haploinsufficient in 48% of RAEB as well as low-risk MDS cases with del(5q). Overall, defective G3BP1 is associated with shorter overall survival (P<.001) in AML, consistent with the reports that del(5q) is a worse prognostic factor in myeloid neoplasms with aggressive phenotype. G3BP1 is a nuclear RNA-binding protein and is ubiquitously expressed in bone marrow, CD34+ progenitors and leukemic cell lines. Furthermore, G3BP1 binds to TP53 and its expression leads to the redistribution of TP53 from the nucleus to the cytoplasm. Similar to RPS14, haploinsufficient of G3BP1 resulted in TP53 up-modulation. Moreover, low expression of G3BP1 in diploid 5q cases was indeed associated with higher TP53 expression. Next, we generated haploinsufficient G3BP1 cell lines using shRNA transduction. Decreased expression of G3BP1 led to growth inhibition and impaired colony formation by transduced cells lines and hematopoietic progenitor cells, respectively. Knockdown of G3BP1 in K562 cell line increased TP53 in the nucleus, and when treated with CPT11, DNA-damaged induced G1-arrest was more prominent in knockdown cells. Furthermore, after knockdown of G3BP1 in TP53-null HL60 cells, we observed increased aneuploidy, suggesting that the loss of function of G3BP1 and TP53 may result in chromosomal instability. Most significantly, G3bp1-/+ mice showed lower blood counts and defective, dysplastic hematopoiesis, similar to lower-risk MDS. As previously described, TP53 defects are associated with advanced disease but recently it became apparent that TP53 may be one of the most common somatic lesions found in the context of del(5q). We stipulate that loss of TP53 function might overcome TP53 tumor suppressor effects and induce leukemic evolution in the defective G3BP1 status. In our cohort, TP53 mutations were more frequently present in high-risk phenotype with G3BP1 haploinsufficient expression. In conclusion, novel somatic mutations of G3BP1 suggest that it could be a candidate gene associated with the clonal evolution of del(5q). Loss of function or low expression of G3BP1 has been shown to up-modulate TP53 and result in dysplasia and growth inhibition, hallmarks of early stages of MDS. Additional events constitute loss of function of TP53, resulting in chromosomal instability, which is associated with leukemogenesis. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.
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Liu, Zhixin, Tao Rui, Zhaoyu Lin, Shule Xie, Bin Zhou, Min Fu, Lianxi Mai, Chuandong Zhu, Guotao Wu, and Youyuan Wang. "Tumor-Associated Macrophages Promote Metastasis of Oral Squamous Cell Carcinoma via CCL13 Regulated by Stress Granule." Cancers 14, no. 20 (October 17, 2022): 5081. http://dx.doi.org/10.3390/cancers14205081.
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M2 tumor-associated macrophages (TAMs) have been a well-established promoter of oral squamous cell carcinoma (OSCC) progression. However, the mechanisms of M2 TAMs promoting OSCC metastasis have not been elucidated clearly. This study illustrated the regulatory mechanisms in which M2 TAMs enhance OSCC malignancy in a novel point of view. In this study, mass spectrometry was utilized to analyze the proteins expression profile of M2 type monocyte-derived macrophages (MDMs-M2), whose results revealed the high expression of G3BP1 in M2 macrophages. RNA sequencing analyzed the genome-wide changes upon G3BP1 knockdown in MDMs-M2 and identified that CCL13 was the most significantly downregulated inflammatory cytokines in MDMs-M2. Co-immunoprecipitation and qualitative mass spectrometry were used to identify the proteins that directly interacted with endogenous G3BP1 in MDMs-M2. Elevated stress granule (SG) formation in stressed M2 TAMs enhanced the expression of CCL13, which promoted OSCC metastasis both in vitro and in vivo. For mechanisms, we demonstrated SG formation improved DDX3Y/hnRNPF-mediated CCL13 mRNA stability, thus enhancing CCL13 expression and promoting OSCC metastasis. Collectively, our findings demonstrated for the first time the roles of CCL13 in improving OSCC metastasis and illustrated the molecular mechanisms of CCL13 expression regulated by SG, indicating that the SG-CCL13 axis can be the potential targets for TAM-navigated tumor therapy.
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Kirchhof, Luisa, Youssef Fouani, Andrea Knau, Galip S. Aslan, Andreas W. Heumüller, Ilka Wittig, Michaela Müller-McNicoll, Stefanie Dimmeler, and Nicolas Jaé. "The G3BP1-UPF1-Associated Long Non-Coding RNA CALA Regulates RNA Turnover in the Cytoplasm." Non-Coding RNA 8, no. 4 (June 30, 2022): 49. http://dx.doi.org/10.3390/ncrna8040049.
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Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.
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He, Xiaomei, Jun Yuan, and Yinsheng Wang. "G3BP1 binds to guanine quadruplexes in mRNAs to modulate their stabilities." Nucleic Acids Research 49, no. 19 (October 6, 2021): 11323–36. http://dx.doi.org/10.1093/nar/gkab873.
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Abstract RNA guanine quadruplexes (rG4) assume important roles in post-transcriptional regulations of gene expression, which are often modulated by rG4-binding proteins. Hence, understanding the biological functions of rG4s requires the identification and functional characterizations of rG4-recognition proteins. By employing a bioinformatic approach based on the analysis of overlap between peaks obtained from rG4-seq analysis and those detected in >230 eCLIP-seq datasets for RNA-binding proteins generated from the ENCODE project, we identified a large number of candidate rG4-binding proteins. We showed that one of these proteins, G3BP1, is able to bind directly to rG4 structures with high affinity and selectivity, where the binding entails its C-terminal RGG domain and is further enhanced by its RRM domain. Additionally, our seCLIP-Seq data revealed that pyridostatin, a small-molecule rG4 ligand, could displace G3BP1 from mRNA in cells, with the most pronounced effects being observed for the 3′-untranslated regions (3′-UTR) of mRNAs. Moreover, luciferase reporter assay results showed that G3BP1 positively regulates mRNA stability through its binding with rG4 structures. Together, we identified a number of candidate rG4-binding proteins and validated that G3BP1 can bind directly with rG4 structures and regulate the stabilities of mRNAs.
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Jayabalan, Aravinth Kumar, Srivathsan Adivarahan, Aakash Koppula, Rachy Abraham, Mona Batish, Daniel Zenklusen, Diane E. Griffin, and Anthony K. L. Leung. "Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity." Proceedings of the National Academy of Sciences 118, no. 6 (February 5, 2021): e2021719118. http://dx.doi.org/10.1073/pnas.2021719118.
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While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)—a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase–deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.
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Wang, Chao, Qiqi Cui, Runxuan Du, Shuang Liu, Shaoping Tian, Hua Huang, Yihua Jiang, et al. "Expression of G3BP1 in benign and malignant human prostate tissues." Translational Andrology and Urology 10, no. 4 (April 2021): 1665–75. http://dx.doi.org/10.21037/tau-20-1450.
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Motaln, Helena, Urša Čerček, Nina Recek, Ana Bajc Česnik, Miran Mozetič, and Boris Rogelj. "Cold atmospheric plasma induces stress granule formation via an eIF2α-dependent pathway." Biomaterials Science 8, no. 19 (2020): 5293–305. http://dx.doi.org/10.1039/d0bm00488j.
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Cold atmospheric plasma treatment of FlpIn SH-SY5Y cells with an inducible expression of G3BP1 results in stress granule assembly resembling a cellular oxidative stress response that has been shown to be eIF2α-signaling dependent and inhibited by an ISRIB inhibitor.
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Herman, Allison B., Milessa Silva Afonso, Sheri E. Kelemen, Mitali Ray, Christine N. Vrakas, Amy C. Burke, Rosario G. Scalia, Kathryn Moore, and Michael V. Autieri. "Regulation of Stress Granule Formation by Inflammation, Vascular Injury, and Atherosclerosis." Arteriosclerosis, Thrombosis, and Vascular Biology 39, no. 10 (October 2019): 2014–27. http://dx.doi.org/10.1161/atvbaha.119.313034.
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Objective: Stress granules (SGs) are dynamic cytoplasmic aggregates containing mRNA, RNA-binding proteins, and translation factors that form in response to cellular stress. SGs have been shown to contribute to the pathogenesis of several human diseases, but their role in vascular diseases is unknown. This study shows that SGs accumulate in vascular smooth muscle cells (VSMCs) and macrophages during atherosclerosis. Approach and Results: Immunohistochemical analysis of atherosclerotic plaques from LDLR − /− mice revealed an increase in the stress granule-specific markers Ras-G3BP1 (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP1+ SGs were rapidly induced in VSMC and bone marrow–derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α (eukaryotic translation initiation factor 2-alpha) phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes. Conclusions: These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.
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Fernandes, Nikita, Luke Nero, Shawn M. Lyons, Pavel Ivanov, Telsa M. Mittelmeier, Timothy A. Bolger, and J. Ross Buchan. "Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation." Biomolecules 10, no. 10 (September 25, 2020): 1367. http://dx.doi.org/10.3390/biom10101367.
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Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Interestingly, in SG assembly mutant cells (G3BP1/2ΔΔ), TDP-43 is enriched in nucleoli. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in G3BP1/2ΔΔ cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events.
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Sun, Zhen, Songlei Xue, Meiying Zhang, Hui Xu, Xuming Hu, Shihao Chen, Yangyang Liu, Mingzhou Guo, and Hengmi Cui. "Aberrant NSUN2-mediated m5C modification of H19 lncRNA is associated with poor differentiation of hepatocellular carcinoma." Oncogene 39, no. 45 (September 25, 2020): 6906–19. http://dx.doi.org/10.1038/s41388-020-01475-w.
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Abstract RNA methylation is an important epigenetic modification. Recent studies on RNA methylation mainly focus on the m6A modification of mRNA, but very little is known about the m5C modification. NSUN2 is an RNA methyltransferase responsible for the m5C modification of multiple RNAs. In this study, we knocked down the NSUN2 gene in HepG2 cells by CRISPR/Cas9 technology and performed high-throughput RNA-BisSeq. An important tumor-related lncRNA H19 was identified to be targeted by NSUN2. Studies have shown that the expression of H19 lncRNA is abnormally elevated and has a carcinogenic effect in many types of tumors. Our results demonstrated that m5C modification of H19 lncRNA can increase its stability. Interestingly, m5C-modified H19 lncRNA can be specifically bound by G3BP1, a well-known oncoprotein which further leads to MYC accumulation. This may be a novel mechanism by which lncRNA H19 exerts its oncogenic effect. Besides, both the m5C methylation level and the expression level of H19 lncRNA in hepatocellular carcinoma tissues were significantly higher than those in adjacent non-cancer tissues, which were closely associated with poor differentiation of hepatocellular carcinoma (HCC). In conclusion, we found that H19 RNA is a specific target for the NSUN2 modifier. The m5C-modified H19 lncRNA may promote the occurrence and development of tumors by recruiting the G3BP1 oncoprotein. Our findings may provide a potential target and biomarker for the diagnosis and treatment of HCC.
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Schneider-Lunitz, Valentin, Jorge Ruiz-Orera, Norbert Hubner, and Sebastiaan van Heesch. "Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes." PLOS Computational Biology 17, no. 12 (December 8, 2021): e1009658. http://dx.doi.org/10.1371/journal.pcbi.1009658.
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RNA-binding proteins (RBPs) can regulate more than a single aspect of RNA metabolism. We searched for such previously undiscovered multifunctionality within a set of 143 RBPs, by defining the predictive value of RBP abundance for the transcription and translation levels of known RBP target genes across 80 human hearts. This led us to newly associate 27 RBPs with cardiac translational regulation in vivo. Of these, 21 impacted both RNA expression and translation, albeit for virtually independent sets of target genes. We highlight a subset of these, including G3BP1, PUM1, UCHL5, and DDX3X, where dual regulation is achieved through differential affinity for target length, by which separate biological processes are controlled. Like the RNA helicase DDX3X, the known splicing factors EFTUD2 and PRPF8—all identified as multifunctional RBPs by our analysis—selectively influence target translation rates depending on 5’ UTR structure. Our analyses identify dozens of RBPs as being multifunctional and pinpoint potential novel regulators of translation, postulating unanticipated complexity of protein-RNA interactions at consecutive stages of gene expression.
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Rao, Lang, Yi Xu, Lucas Charles Reineke, Abhisek Bhattacharya, Alexey Tyryshkin, Jin Na Shin, and N. Tony Eissa. "Post-Transcriptional Regulation of Alpha One Antitrypsin by a Proteasome Inhibitor." International Journal of Molecular Sciences 21, no. 12 (June 17, 2020): 4318. http://dx.doi.org/10.3390/ijms21124318.
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Alpha one antitrypsin (α1AT), a serine proteinase inhibitor primarily produced by the liver, protects pulmonary tissue from neutrophil elastase digestion. Mutations of the SERPINA1 gene results in a misfolded α1AT protein which aggregates inside hepatocytes causing cellular damage. Therefore, inhibition of mutant α1AT production is one practical strategy to alleviate liver damage. Here we show that proteasome inhibitors can selectively downregulate α1AT expression in human hepatocytes by suppressing the translation of α1AT. Translational suppression of α1AT is mediated by phosphorylation of eukaryotic translation initiation factor 2α and increased association of RNA binding proteins, especially stress granule protein Ras GAP SH3 binding protein (G3BP1), with α1AT mRNA. Treatment of human-induced pluripotent stem cell-derived hepatocytes with a proteasome inhibitor also results in translational inhibition of mutant α1AT in a similar manner. Together we revealed a previously undocumented role of proteasome inhibitors in the regulation of α1AT translation.
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Deng, Wei, Mingxia Yan, Tao Yu, Haiyan Ge, Hechun Lin, Jing Li, Ying Liu, et al. "Quantitative Proteomic Analysis of the Metastasis-Inhibitory Mechanism of miR-193a-3p in Non-Small Cell Lung Cancer." Cellular Physiology and Biochemistry 35, no. 5 (2015): 1677–88. http://dx.doi.org/10.1159/000373981.
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Background: microRNAs can repress the expression of target genes by destabilizing their mRNAs or by inhibiting their translation. Our previous findings suggested that miR-193a-3p inhibited the progression of NSCLC both in vitro and in vivo. However, the biological processes and molecular pathways through which this miRNA exerts its positive effects are unknown. Methods: To explore the molecular mechanisms by which miR-193a-3p inhibited NSCLC metastasis, we investigated the changes in the protein profile of SPC-A-1sci (highly metastatic) cells in response to the up-regulation of miR-193a-3p expression using a proteomics approach (iTRAQ combined with NanoLC-MS/MS). Changes in the profiles of the expressed proteins were verified using western blotting and were analyzed using the DAVID and STRING programs. Results: In the two replicated experiments, 4962/4946 proteins were identified, and the levels of expression of 4923/4902 proteins were quantified. In total, 112 of these proteins were differentially expressed. Among them, the up-regulated levels of expression of two of the 62 proteins with up-regulated expression (PPP2R2A and GSN) and the down-regulated levels of expression four of the 50 proteins with down-regulated expression (LMNB2, UHRF1, G3BP1, and HNRNPU) were verified using western blotting. The bioinformatics analysis revealed the interactions and signaling networks of these differentially expressed proteins. Conclusion: miR-193a-3p inhibited the metastasis of lung cancer cells by deregulating the expression of tumor-related proteins. These findings may improve the understanding of the molecular mechanisms underlying the metastatic-inhibitory effect of miR-193a-3p on lung cancer cells.
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Ying, Shan, and Denys A. Khaperskyy. "UV damage induces G3BP1-dependent stress granule formation that is not driven by mTOR inhibition-mediated translation arrest." Journal of Cell Science 133, no. 20 (September 28, 2020): jcs248310. http://dx.doi.org/10.1242/jcs.248310.
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ABSTRACTTranslation arrest is a part of the cellular stress response that decreases energy consumption and enables rapid reprioritisation of gene expression. Often translation arrest leads to condensation of untranslated messenger ribonucleoproteins (mRNPs) into stress granules (SGs). Studies into mechanisms of SG formation and functions are complicated because various types of stress cause formation of SGs with different properties and composition. In this work, we focused on the mechanism of SG formation triggered by UV damage. We demonstrate that UV-induced inhibition of translation does not involve inhibition of the mechanistic target of rapamycin (mTOR) signaling or dissociation of the 48S preinitiation complexes. The general control non-derepressible 2 (GCN2; also known as EIF2AK4) kinase contributes to UV-induced SG formation, which is independent of the phosphorylation of the eukaryotic translation initiation factor 2α. Like many other types of SGs, condensation of UV-induced granules requires the Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1). Our work reveals that, in UV-treated cells, the mechanisms of translation arrest and SG formation may be unlinked, resulting in SGs that do not contain the major type of polysome-free preinitiation complexes that accumulate in the cytoplasm.This article has an associated First Person interview with the first author of the paper.
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Schöbel, Anja, Van Nguyen-Dinh, Gerald G. Schumann, and Eva Herker. "Hepatitis C virus infection restricts human LINE-1 retrotransposition in hepatoma cells." PLOS Pathogens 17, no. 4 (April 19, 2021): e1009496. http://dx.doi.org/10.1371/journal.ppat.1009496.
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LINE-1 (L1) retrotransposons are autonomous transposable elements that can affect gene expression and genome integrity. Potential consequences of exogenous viral infections for L1 activity have not been studied to date. Here, we report that hepatitis C virus (HCV) infection causes a significant increase of endogenous L1-encoded ORF1 protein (L1ORF1p) levels and translocation of L1ORF1p to HCV assembly sites at lipid droplets. HCV replication interferes with retrotransposition of engineered L1 reporter elements, which correlates with HCV RNA-induced formation of stress granules and can be partially rescued by knockdown of the stress granule protein G3BP1. Upon HCV infection, L1ORF1p localizes to stress granules, associates with HCV core in an RNA-dependent manner and translocates to lipid droplets. While HCV infection has a negative effect on L1 mobilization, L1ORF1p neither restricts nor promotes HCV infection. In summary, our data demonstrate that HCV infection causes an increase of endogenous L1 protein levels and that the observed restriction of retrotransposition of engineered L1 reporter elements is caused by sequestration of L1ORF1p in HCV-induced stress granules.
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Lee, Saebom, Hye Guk Ryu, Sin Ho Kweon, Hyerynn Kim, Hyeonwoo Park, Kyung-Ha Lee, Sang-Min Jang, Chan Hyun Na, Sangjune Kim, and Han Seok Ko. "c-Abl Regulates the Pathological Deposition of TDP-43 via Tyrosine 43 Phosphorylation." Cells 11, no. 24 (December 8, 2022): 3972. http://dx.doi.org/10.3390/cells11243972.
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Non-receptor tyrosine kinase, c-Abl plays a role in the pathogenesis of several neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Here, we found that TDP-43, which was one of the main proteins comprising pathological deposits in amyotrophic lateral sclerosis (ALS), is a novel substrate for c-Abl. The phosphorylation of tyrosine 43 of TDP-43 by c-Abl led to increased TDP-43 levels in the cytoplasm and increased the formation of G3BP1-positive stress granules in SH-SY5Y cells. The kinase-dead mutant of c-Abl had no effect on the cytoplasmic localization of TDP-43. The expression of phosphor-mimetic mutant Y43E of TDP-43 in primary cortical neurons accumulated the neurite granule. Furthermore, the phosphorylation of TDP-43 at tyrosine 43 by c-Abl promoted the aggregation of TDP-43 and increased neuronal cell death in primary cortical neurons, but not in c-Abl–deficient primary cortical neurons. Identification of c-Abl as the kinase of TDP43 provides new insight into the pathogenesis of ALS.
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Zheng, Hongmei, Yuting Zhan, Yuting Zhang, Sile Liu, Junmi Lu, Yang yang, Qiuyuan Wen, and Songqing Fan. "Elevated expression of G3BP1 associates with YB1 and p‐AKT and predicts poor prognosis in nonsmall cell lung cancer patients after surgical resection." Cancer Medicine 8, no. 16 (September 27, 2019): 6894–903. http://dx.doi.org/10.1002/cam4.2579.
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Campbell, Taaliah, Ohuod Hawsawi, Nathan Bowen, and Valerie Odero-Marah. "Abstract 113: Investigating the role of high mobility group a2 (HMGA2) truncated isoform in promoting oxidative stress in PCa cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 113. http://dx.doi.org/10.1158/1538-7445.am2022-113.
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Abstract Prostate cancer (PCa) is one of the most commonly diagnosed malignancies among men worldwide and remains the second leading cause of cancer related death in the United States. Oxidative stress has been shown to be increase in several cancers including prostate cancer. In fact, oxidative stress in prostate cancer is suggested to be a direct result of cell exposure to reactive oxygen species (ROS). High mobility group A 2 (HMGA2), a non-histone protein, is an oncogene that is up-regulated in several cancers. This protein has ability to undergo chromosomal rearrangement and alternative splicing, causing its full length/wild type HMGA2 (HMGA2-WT) to become the truncated losing its 3’UTR leading to the generation of HMGA2 truncated (HMGA2-TR). We have previously shown HMGA2-WT’s involvement in epithelial mesenchymal transition (EMT), however, the functional role of HMGA2-TR has not yet been dissected. We hypothesize that truncated HMGA2’s involvement with oxidative stress leads to prostate cancer progression. We analyzed the baseline expression of wild-type vs.truncated HMGA2 in prostate patient tissue and cells lines by real-time PCR and western blot analyses. Prostate cancer patient tissue and some cell lines expressed increasing amounts of both wild-type and truncated HMGA2with increasing tumor grade, when compared to normal epithelial cells. RNA-Seq analysis of LNCaP prostate cancer cells stably overexpressing HMGA2-WT, HMGA2-TR, or empty vector (Neo) control revealed thatHMGA2-TR cells display increased oxidative stress compared to HMGA2-WT or Neo control cells. This was also confirmed by analysis of basal reactive oxygen species (ROS) levels, and the ratio of GSH/GSSG andNADP/NADPH utilizing metabolomics. Additionally, proteomic analysis showed that HMGA2-TR protein interacted with several proteins, including a cytoplasmic stress granule protein G3BP1 that responds to oxidative stress. Transient knockdown of G3BP1 increased ROS in HMGA2-TR cells. These studies may therefore uncover novel role for truncated HMGA2 in oxidative stress. Acknowledgements: These studies were supported by the NIH/NIMHD/RCI Grant #5G12MD007590-31,NIH/NIGMS/RISE Grant #5R25GM060414 Citation Format: Taaliah Campbell, Ohuod Hawsawi, Nathan Bowen, Valerie Odero-Marah. Investigating the role of high mobility group a2 (HMGA2) truncated isoform in promoting oxidative stress in PCa cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 113.
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Thiel, Cora Sandra, Swantje Christoffel, Svantje Tauber, Christian Vahlensieck, Diane de Zélicourt, Liliana E. Layer, Beatrice Lauber, Jennifer Polzer, and Oliver Ullrich. "Rapid Cellular Perception of Gravitational Forces in Human Jurkat T Cells and Transduction into Gene Expression Regulation." International Journal of Molecular Sciences 21, no. 2 (January 14, 2020): 514. http://dx.doi.org/10.3390/ijms21020514.
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Cellular processes are influenced in many ways by changes in gravitational force. In previous studies, we were able to demonstrate, in various cellular systems and research platforms that reactions and adaptation processes occur very rapidly after the onset of altered gravity. In this study we systematically compared differentially expressed gene transcript clusters (TCs) in human Jurkat T cells in microgravity provided by a suborbital ballistic rocket with vector-averaged gravity (vag) provided by a 2D clinostat. Additionally, we included 9× g centrifuge experiments and rigorous controls for excluding other factors of influence than gravity. We found that 11 TCs were significantly altered in 5 min of flight-induced and vector-averaged gravity. Among the annotated clusters were G3BP1, KPNB1, NUDT3, SFT2D2, and POMK. Our results revealed that less than 1% of all examined TCs show the same response in vag and flight-induced microgravity, while 38% of differentially regulated TCs identified during the hypergravity phase of the suborbital ballistic rocket flight could be verified with a 9× g ground centrifuge. In the 2D clinostat system, doing one full rotation per second, vector effects of the gravitational force are only nullified if the sensing mechanism requires 1 s or longer. Due to the fact that vag with an integration period of 1 s was not able to reproduce the results obtained in flight-induced microgravity, we conclude that the initial trigger of gene expression response to microgravity requires less than 1 s reaction time. Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces during cell culture experiments in general.
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Bomberger, Jennifer, Kenneth Ely, Naveen Bangia, Brent Berwin, William Green, Richard Enelow, and Bruce Stanton. "Pseudomonas aeruginosa reduces the immune response to respiratory viruses (99.33)." Journal of Immunology 186, no. 1_Supplement (April 1, 2011): 99.33. http://dx.doi.org/10.4049/jimmunol.186.supp.99.33.
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Abstract P. aeruginosa chronically colonizes the respiratory tract of patients with chronic obstructive pulmonary disease, bronchiectasis and Cystic Fibrosis. These patients experience debilitating and life-threatening viral exacerbations of unknown etiology. Cif, a bacterial toxin secreted by P. aeruginosa, promotes the degradation of several ABC transporters involved in innate immunity, including CFTR and Pgp. The current study tests the hypothesis that Cif enhances the degradation of the transporter associated with antigen processing (TAP), resulting in reduced adaptive T-cell responses to viral pathogens. Cif inhibited the deubiquitinating enzyme, ubiquitin specific protease 10 (USP10), resulting in a time-dependent increase in the polyubiquitination and proteasomal degradation of TAP1. Cif inhibited USP10 activity by stabilizing an inhibitory protein-protein interaction between USP10 and G3BP1. The Cif-mediated decrease in TAP1 expression resulted in decreased MHC class I viral antigen presentation, CD8+ cytotoxic T lymphocyte (CTL) recognition of influenza virus-infected cells and dramatically reduced MHC class I cell surface levels on CD11c+ cells in the BAL of mice treated with P. aeruginosa. We propose that P. aeruginosa, by secreting the Cif toxin, inhibits the ability of CTL to recognize and eliminate viral infections, severely compromising the immune defenses of the lung. This is the first identified bacterial toxin that inhibits the viral immune response of the host.
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Bai, Zhile, Mengyu Feng, Yang Du, Lin Cong, and Yong Cheng. "Carboxypeptidase E down-regulation regulates transcriptional and epigenetic profiles in pancreatic cancer cell line: A network analysis." Cancer Biomarkers 29, no. 1 (September 25, 2020): 79–88. http://dx.doi.org/10.3233/cbm-191163.
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BACKGROUND: Pancreatic cancer is a malignant tumor and its incidence has increased in recent years. Carboxypeptidase E (CPE) is a prohormone/proneuropeptide processing enzyme that has been shown to be associated with tumor growth and invasion in various cancers including pancreatic cancer. OBJECTIVE: To understand the molecular mechanism underlying the proliferative effects of CPE in cancer cells. METHODS: We down-regulated CPE gene expression in PANC-1 cell, a pancreatic cell line, and investigated mRNA, miRNA, circRNA and lncRNA expression profiling in PANC-1 cells from control group and CPE knock-down group by microarray analysis. We further validated the top 14 differentially expressed circRNAs by qRT-PCR. RESULTS: Our results showed that CPE down-regulation caused decreased cell proliferation. The microarray data showed 107, 15, 299 and 360 differentially expressed mRNAs, miRNAs, circRNAs, and lncRNAs, respectively between control group and CPE knock-down group. Of Which, 41 mRNAs, 12 miRNAs, 133 circRNAs, and 262 lncRNAs were down-regulated; 66 mRNAs, 3 miRNAs, 166 circRNAs, and 98 lncRNAs were up-regulated. Bioinformatics analysis showed that the top significantly enriched pathways for the differentially expressed RNAs were related to cancer onset and/or progression, these included p53 signaling pathway, ECM-receptor interaction, focal adhesion and Wnt signaling pathway. We further performed network analysis to assess the mRNA, miRNA, circRNA and lncRNA correlations, and showed that HUWE1, hsa-miR-6780b-5p, has_circ_0058208 and lnc-G3BP1-3:8 were in the core position of the network. CONCLUSIONS: Taken together, these results identified potential CPE regulated core genes and pathways for cell proliferation in pancreatic cancer cell, and therefore provide potential targets for the treatment of pancreatic cancer.
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Campbell, Ashley M., Carlos F. De La Cruz-Herrera, Edyta Marcon, Jack Greenblatt, and Lori Frappier. "Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function." PLOS Pathogens 18, no. 1 (January 10, 2022): e1010235. http://dx.doi.org/10.1371/journal.ppat.1010235.
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The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3’UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3’UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.
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Zhao, Zhicong, Lei Dong, Li Han, Yangchan Li, Jianhuang Xue, Keren Zhou, Wei Li, et al. "Abstract 5685: Recognition of internal mRNA N7-methylguanosine by QKI shuttles transcripts into stress granules and modulates drug resistance." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5685. http://dx.doi.org/10.1158/1538-7445.am2022-5685.
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Abstract Introduction: Albeit that over 170 types of chemical modifications have been identified in RNAs, the biological functions of most of those decorations are still elusive. N7-methylguanosine (m7G), routinely occurring at the 5’ cap of mRNA or within tRNA and rRNA, also exists in internal mRNA. However, the “reader” proteins that recognize internal mRNA m7G and regulate the metabolism and fate of target mRNAs have yet to be identified. Here, we aim to identify the reader(s) of internal mRNA m7G and elucidate the biological function. Methods: To identify internal m7G reader(s), we utilized RNA-pull down, mass spectrometry (MS), RNA immunoprecipitation (RIP)-seq, and m7G-seq. To evaluate the interaction between QKI and G3BP1, we used Co-IP, immunofluorescence (IF) and proximity ligation assay (PLA). To investigate the function of QKI in stress granule (SG), we performed polyA & m7G RNA fluorescence in situ hybridization (FISH), triple quadrupole MS (QQQ-MS), and Ribo-seq. To assess the role of QKI in drug resistance, we carried out both in vitro cell survival assays and in vivo xenograft mouse model. Results: Our QQQ-MS results suggest cytoplasmic mRNA, in contrast to nuclei mRNA, are highly enriched with internal m7G modification. Quaking proteins (QKI), especially QKI7, can selectively recognize the internal mRNA m7G decoration in the cytosol of various cell types. Our high-throughput sequence data revealed over 1000 confident m7G-modified and QKI-binding RNA targets with a conserved motif, “GANGAN (N=A/U/G)”. Those transcripts are functionally enriched in the “pathways in cancer” and “Hippo signaling pathway”. More strikingly, internal m7G reader QKI7 directly interacts with the SG core protein G3BP1 and can shuttle a subset of m7G-modified transcripts into SG mRNA pool under oxidative stress condition. Additionally, we identified 314 confident m7G-modified & QKI7-binding & SG-enriched target genes in U2OS cells. Ribo-seq indicated that QKI7 modulates the translation efficiency of a set of m7G-modified transcripts via sequestering and silencing them in SGs. Moreover, in line with the observation that many chemotherapy drugs could trigger the assembly of SGs, QKI7 increases the sensitivity of cancer cells to drug treatment (e.g., doxorubicin) in vitro and in vivo in a m7G-dependent manner. Consistently, by analyzing the hepatocellular carcinoma (HCC) pharmacogenomic landscape study dataset, we found that HCC cell lines with higher QKI expression levels are more sensitive to a variety of chemotherapy or targeted therapeutic drugs. Conclusion: Our study identifies QKI as the first internal mRNA m7G reader, provides insights into the biological function of internal mRNA m7G modification in remodeling stress granules transcriptome, and highlights the therapeutic potential of targeting the QKI7/m7G axis in overcoming drug resistance. Citation Format: Zhicong Zhao, Lei Dong, Li Han, Yangchan Li, Jianhuang Xue, Keren Zhou, Wei Li, Xiaolan Deng, Ying Qing, Brandon Tan, Zhenhua Chen, Chao Shen, Andrew Small, Kitty Wang, Keith Leung, Zheng Zhang, Xi Qin, Qiang Xia, Rui Su, Jianjun Chen. Recognition of internal mRNA N7-methylguanosine by QKI shuttles transcripts into stress granules and modulates drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5685.
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Jahan, Sadaf, Uzair Ahmad Ansari, Arif Jamal Siddiqui, Danish Iqbal, Johra Khan, Saeed Banawas, Bader Alshehri, et al. "Nobiletin Ameliorates Cellular Damage and Stress Response and Restores Neuronal Identity Altered by Sodium Arsenate Exposure in Human iPSCs-Derived hNPCs." Pharmaceuticals 15, no. 5 (May 12, 2022): 593. http://dx.doi.org/10.3390/ph15050593.
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Environmental exposure to arsenic has been profoundly associated with chronic systemic disorders, such as neurodegeneration, in both experimental models and clinical studies. The neuronal cells of the brain and the nervous system have a limited regeneration capacity, thus making them more vulnerable to exposure to xenobiotics, leading to long-lasting disabilities. The functional and anatomical complexity of these cells hinders the complete understanding of the mechanisms of neurodegeneration and neuroprotection. The present investigations aimed to evaluate the neuroprotective efficacy of a herbal formulation of Nobiletin (NOB) against the toxic insult induced by sodium arsenate (NA) in human neural progenitor cells (hNPCs) derived from human induced pluripotent stem cells (hiPSCs). Prior to the neuroprotective experiments, biologically safe doses of both NOB and NA were ascertained using standard endpoints of cytotoxicity. Thereafter, the hNPCs were exposed to either NOB (50 μM) or NA (50 μM) and co-exposed to biologically safe concentrations of NA (50 μM) with NOB (50 μM) for a period of up to 48 h. NOB treatment restored the morphological damage (neurite damage), the levels of stress granule G3BP1 (Ras-GTPase-activating protein (SH3 domain)-binding protein) and TIA1 (T cell-restricted intracellular antigen), and the expression of neuronal markers (Tuj1, Nestin, MAP2, and PAX6) when compared to NA-exposed cells. A substantial restoration of reactive oxygen species and mitochondrial membrane potential was also witnessed in the co-exposure group (NA + NOB) in comparison to the NA-exposed group. The findings suggest that NOB possesses a significant restorative/protective potential against the NA challenge in hNPCs under experimental conditions and imply that nobiletin may impart a potential therapeutic impact if studied adequately using in vivo studies.
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Cristea, Ileana M., Heather Rozjabek, Kelly R. Molloy, Sophiya Karki, Laura L. White, Charles M. Rice, Michael P. Rout, Brian T. Chait, and Margaret R. MacDonald. "Host Factors Associated with the Sindbis Virus RNA-Dependent RNA Polymerase: Role for G3BP1 and G3BP2 in Virus Replication." Journal of Virology 84, no. 13 (April 14, 2010): 6720–32. http://dx.doi.org/10.1128/jvi.01983-09.
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ABSTRACT Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3×Flag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
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Zheng, Yinli, Jinjun Wu, Ru Deng, Censhan Lin, Yuhua Huang, Xia Yang, Chunhua Wang, et al. "G3BP2 regulated by the lncRNA LINC01554 facilitates esophageal squamous cell carcinoma metastasis through stabilizing HDGF transcript." Oncogene 41, no. 4 (November 15, 2021): 515–26. http://dx.doi.org/10.1038/s41388-021-02073-0.
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AbstractMetastasis is the leading cause of death of patients with esophageal squamous cell carcinoma (ESCC). Although an increasing number of studies have demonstrated the involvement of G3BP2 in several human cancers, how G3BP2 interacts with long noncoding RNAs and regulates mRNA transcripts in mediating ESCC metastasis remains unclear. In this study, we uncovered that G3BP2 was upregulated in ESCC. Further analysis revealed that upregulation of G3BP2 was significantly correlated with lymph node metastasis, depth of tumor invasion and unfavorable outcomes in ESCC patients. Both in vitro and in vivo functional assays demonstrated that G3BP2 dramatically enhanced ESCC cell migration and invasion. Mechanistically, LINC01554 maintained the high G3BP2 expression in ESCC by protecting G3BP2 from degradation through ubiquitination and the interaction domains within LINC01554 and G3BP2 were identified. In addition, RNA-seq revealed that HDGF was regulated by G3BP2. G3BP2 bound to HDGF mRNA transcript to stabilize its expression. Ectopic expression of HDGF effectively abolished the G3BP2 depletion-mediated inhibitory effect on tumor cell migration. Intriguingly, introduction of compound C108 which can inhibit G3BP2 remarkedly suppressed ESCC cell metastasis in vitro and in vivo. Collectively, this study describes a newly discovered regulatory axis, LINC01554/G3BP2/HDGF, that facilitates ESCC metastasis and will provide novel therapeutic strategies for ESCC.
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Zekri, Latifa, Karim Chebli, Hélène Tourrière, Finn C. Nielsen, Thomas V. O. Hansen, Abdelhaq Rami, and Jamal Tazi. "Control of Fetal Growth and Neonatal Survival by the RasGAP-Associated Endoribonuclease G3BP." Molecular and Cellular Biology 25, no. 19 (October 1, 2005): 8703–16. http://dx.doi.org/10.1128/mcb.25.19.8703-8716.2005.
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ABSTRACT The regulation of mRNA stability plays a major role in the control of gene expression during cell proliferation, differentiation, and development. Here, we show that inactivation of the RasGAP-associated endoribonuclease (G3BP)-encoding gene leads to embryonic lethality and growth retardation. G3BP − / − mice that survived to term exhibited increased apoptotic cell death in the central nervous system and neonatal lethality. Both in mouse embryonic fibroblasts and during development, the absence of G3BP altered the expression of essential growth factors, among which imprinted gene products and growth arrest-specific mRNAs were outstanding. The results demonstrate that G3BP is essential for proper embryonic growth and development by mediating the coordinate expression of multiple imprinted growth-regulatory transcripts.
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Hosono, Naoko, Hideki Makishima, Bartlomiej P. Przychodzen, Andres Jerez, Chantana Polprasert, Yuichi Shiraishi, Kenichi Chiba, et al. "Whole Exome Sequencing (“mutatome”) Of Deletion 5q." Blood 122, no. 21 (November 15, 2013): 656. http://dx.doi.org/10.1182/blood.v122.21.656.656.
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Abstract Interstitial deletion of chromosome 5q (del(5q)) is one of the most common karyotypic abnormalities in MDS. While a relatively small fraction of patients with del(5q) and 5q- syndrome show a relatively uniform clinical phenotype and a low rate of progression, the majority of del(5q) myeloid neoplasms are more heterogeneous. Prognosis correlates with the size and location of the deletion, with large deletions spanning subtelomeric and/or subcentromeric region correlating with a poor prognosis. Survival differences may relate to still undefined pathogenetic mechanisms underlying del(5q), which may involve hemizygous mutations or haploinsufficiency. With the latter scenario, it is possible that heterozygous mutations of genes located on 5q may phenocopy the deletion. To further elucidate the molecular mechanisms underlying del(5q), we preformed a comprehensive analysis of myeloid neoplasms using single nucleotide polymorphism array (SNP-A) and next generation whole exome sequencing (NGS) of paired DNA samples (germline/tumor) from 55 cases characterized by del(5q) among a total 428 patients with MDS and related disorders; we focused on mutations located on 5q in both diploid and deletion cases. In the total cohort, we identified 243 somatic mutations in 158 genes on chr5q, including well-known NPM1 or novel recurrent DDX41 mutations; 147 mutations were heterozygous, 11 hemizygous (in del(5q)). No homozygous mutations were found. Applying SNP-A-based karyotyping, we defined the commonly deleted region (CDR) as between 5q32 and 5q33.2 (145299747-153828955). In patients with 5q- syndrome, the proximal and terminal regions of chr5q were always retained; therefore we defined commonly retained regions (CRR) as CRR1 (proximal, 5q11.1 to 5q14.2, 48400001-81634579) and CRR2 (distal, 5q34 to 5q35.3, 164213764-180915260). The deletions of CRRs consequently contributed to worse prognosis in the aggressive types of MDS with longer del(5q). First we focused our study on the genes located on CRRs. We identified 120 heterozygous alterations in CRRs, including CWC27 (5q12.3), MAP1B (n=2, 5q13.2), NPM (n=50, 5q35.1), C5orf25 (n=4, 5q35.2) and DDX41 (n=4, 5q35.3); these mutations occurred only in a heterozygous configuration. Interestingly, spliceosome-associated gene CWC27 and RNA helicase DDX41 showed haploinsufficient expression in haploid cases without mutation, suggesting that mutated genes located on CRRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. Furthermore, patients with decreased expression of these genes had a poor survival (CWC27; HR=2.48, DDX41; HR=1.98). In positions corresponding to CDR and its proximal regions, we found 123 heterozygous alterations in 97 genes (50% of all alterations on 5q found), including recurrently mutated genes (FAT2: n=4; G3BP1: n=2) and hemizygous mutations of KDM3B (n=3, 5q31.2) and MCC (n=1, 5q22.2). In GPR98, associated with Usher syndrome, we detected both recurrent heterozygous and hemizygous mutations (each n=1). Also, minor alleles (frequencies were .002 and .004) of non-synonymous variants of GPR98 were selectively retained and wild-type alleles were deleted in del(5q) cases (n=2). We also searched accessory genetic events observed on other chromosomes in del(5q) cases. By SNP-A, deletions of CRRs (longer del(5q)) were significantly more associated with additional chromosomal defects. Similarly, some specific genes, including the splicing machinery genes and IDH family genes, were uniquely observed in the longer del(5q) cohort. In conclusion, we detected multiple pathogenic mutations in whole chr5q which might stratify del(5q) patients at risk for disease progression, though no single mutations could explain a majority of cases. Decreased expression or mutation of CWC27 and DDX41, located on CRRs, may exemplify the common pathophysiology shared by heterozygous mutations and haploinsufficient expressions on chr5q. Consequently, it is possible that deletion alone, through decreased expression, may be pathogenic. Disclosures: Makishima: AA & MDS international foundation: Research Funding; Scott Hamilton CARES grant: Research Funding. Polprasert:MDS foundation: Research Funding. Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.
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Dolliver, Stacia M., Mariel Kleer, Maxwell P. Bui-Marinos, Shan Ying, Jennifer A. Corcoran, and Denys A. Khaperskyy. "Nsp1 proteins of human coronaviruses HCoV-OC43 and SARS-CoV2 inhibit stress granule formation." PLOS Pathogens 18, no. 12 (December 19, 2022): e1011041. http://dx.doi.org/10.1371/journal.ppat.1011041.
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Stress granules (SGs) are cytoplasmic condensates that often form as part of the cellular antiviral response. Despite the growing interest in understanding the interplay between SGs and other biological condensates and viral replication, the role of SG formation during coronavirus infection remains poorly understood. Several proteins from different coronaviruses have been shown to suppress SG formation upon overexpression, but there are only a handful of studies analyzing SG formation in coronavirus-infected cells. To better understand SG inhibition by coronaviruses, we analyzed SG formation during infection with the human common cold coronavirus OC43 (HCoV-OC43) and the pandemic SARS-CoV2. We did not observe SG induction in infected cells and both viruses inhibited eukaryotic translation initiation factor 2α (eIF2α) phosphorylation and SG formation induced by exogenous stress. Furthermore, in SARS-CoV2 infected cells we observed a sharp decrease in the levels of SG-nucleating protein G3BP1. Ectopic overexpression of nucleocapsid (N) and non-structural protein 1 (Nsp1) from both HCoV-OC43 and SARS-CoV2 inhibited SG formation. The Nsp1 proteins of both viruses inhibited arsenite-induced eIF2α phosphorylation, and the Nsp1 of SARS-CoV2 alone was sufficient to cause a decrease in G3BP1 levels. This phenotype was dependent on the depletion of cytoplasmic mRNA mediated by Nsp1 and associated with nuclear accumulation of the SG-nucleating protein TIAR. To test the role of G3BP1 in coronavirus replication, we infected cells overexpressing EGFP-tagged G3BP1 with HCoV-OC43 and observed a significant decrease in virus replication compared to control cells expressing EGFP. The antiviral role of G3BP1 and the existence of multiple SG suppression mechanisms that are conserved between HCoV-OC43 and SARS-CoV2 suggest that SG formation may represent an important antiviral host defense that coronaviruses target to ensure efficient replication.
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Tourrière, Hélène, Karim Chebli, Latifa Zekri, Brice Courselaud, Jean Marie Blanchard, Edouard Bertrand, and Jamal Tazi. "The RasGAP-associated endoribonuclease G3BP assembles stress granules." Journal of Cell Biology 160, no. 6 (March 17, 2003): 823–31. http://dx.doi.org/10.1083/jcb.200212128.
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Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents, and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149, whose dephosphorylation is induced by arsenite treatment. Critically, a phosphomimetic mutant (S149E) fails to oligomerize and to assemble SGs, whereas a nonphosphorylatable G3BP mutant (S149A) does both. These results suggest that G3BP is an effector of SG assembly, and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.
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Gupta, Nisha, Mark Badeaux, Yiqian Liu, Kamila Naxerova, Dennis Sgroi, Lance L. Munn, Rakesh K. Jain, and Igor Garkavtsev. "Stress granule-associated protein G3BP2 regulates breast tumor initiation." Proceedings of the National Academy of Sciences 114, no. 5 (January 17, 2017): 1033–38. http://dx.doi.org/10.1073/pnas.1525387114.
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Breast tumors contain tumorigenic cancer cells, termed “tumor-initiating cells” (TICs), which are capable of both replenishing themselves and giving rise to populations of nontumorigenic breast cancer cells (non-TICs). However, the molecular mechanisms responsible for breast tumor initiation remain poorly understood. Here we describe a chemical screening strategy to identify small molecules that enhance the effect of chemotherapeutic agents on TIC-enriched breast cancer cells. We identified proteins that interact with the lead compound C108, including the stress granule-associated protein, GTPase-activating protein (SH3 domain)-binding protein 2, G3BP2. G3BP2 regulates breast tumor initiation through the stabilization of Squamous cell carcinoma antigen recognized by T cells 3 (SART3) mRNA, which leads to increased expression of the pluripotency transcription factors Octamer-binding protein 4 (Oct-4) and Nanog Homeobox (Nanog). Our findings suggest that G3BP2 is important for the process of breast cancer initiation. Furthermore, these data suggest a possible connection between stress granule formation and tumor initiation in breast cancer cells.
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Brown, Shelby L., Dana J. Garrison, and Jared P. May. "Phase separation of a plant virus movement protein and cellular factors support virus-host interactions." PLOS Pathogens 17, no. 9 (September 20, 2021): e1009622. http://dx.doi.org/10.1371/journal.ppat.1009622.
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Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.
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Kennedy, Derek, Juliet French, Estelle Guitard, Kelin Ru, Bruno Tocque, and John Mattick. "Characterization of G3BPs: Tissue specific expression, chromosomal localisation andrasGAP120 binding studies." Journal of Cellular Biochemistry 84, no. 1 (November 2, 2001): 173–87. http://dx.doi.org/10.1002/jcb.1277.
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Lehmann, Soren, Sophie Raynaud, Julian C. Desmond, and Phillip H. Koeffler. "Identification of Pathogenetically Important Genes within the Common Deleted Region (CDR) of the 5q- Syndrome." Blood 106, no. 11 (November 16, 2005): 3439. http://dx.doi.org/10.1182/blood.v106.11.3439.3439.
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Abstract The 5q- syndrome is characterized by refractory anemia, normal or high platelet count, hypolobulated megakaryocytes, a good prognosis and a low risk of leukemic transformation. Although the CDR has been defined to a 1.5 Mb interval on the long arm on chromosome 5 (5q33.1), the molecular pathogenesis of the disease is still unknown. The CDR contains 39 known-genes of which 33 have been shown to be expressed in hematopoietic stem cells. In order to elucidate the molecular mechanisms behind the 5q- syndrome, we performed real-time quantitative PCR on these 33 genes. Samples from the bone marrow of 12 patients with a sole deletion of 5q and 14 patients with MDS with normal karyotype were initially analyzed. The genes that showed the most pronounced decrease in expression in the 5q- samples were: SLC36A1 (89% down-regulated compared to non 5q-), G3BP (79%), ATOX1 (76%), CSF1R (76%), RPS14 (74%), PDGFRB (73%), TNIP1 (72%), SPARC (71%), ANAX6 (69%), NSDT (66%) and TIGD (60%). SPARC expression was found to be higher in both types of MDS samples compared to normal bone marrow (n=18) as well as compared to seven leukemic cell lines (HL-60, NB4, HEL, KG1, K562, U937 and TP-1). ATOX1 expression was highly over-expressed (20- to 80-fold) in the leukemic cell lines and modestly but significantly higher in normal bone marrow compared to both types of MDS. For G3BP, the expression was similar in normal bone marrow compared to the non-5q- samples but 1- to 10-fold higher in the cell lines. RPS14 was down-regulated in both types of MDS compared to normal bone marrow and leukemic cell lines. Thus, we have identified the most significantly down-regulated genes within the CDR of the 5q- syndrome. Based on our expression data, their known biological functions and on publicly available tissue expression data, genes such as G3BP, ATOX1, TNIP1, RPS14 and CSF1R are interesting targets for further studies. Biological studies are currently being performed on these genes with respect to their role during hematopoiesis with special focus on erythropoiesis.
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Zhang, Hui-Zhong. "Expression of G3BP and RhoC in esophageal squamous carcinoma and their effect on prognosis." World Journal of Gastroenterology 13, no. 30 (2007): 4126. http://dx.doi.org/10.3748/wjg.v13.i30.4126.
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Basu, Gargi D., Kevin Drenner, Audrey Ozols, Candyce M. Bair, Tracey White, Janine R. LoBello, Thomas Royce, and Sunil Sharma. "Whole exome and transcriptome sequencing of colorectal and pancreatic cancer." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): e15666-e15666. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e15666.
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e15666 Background: Integration of Whole Exome Sequencing (WES) into clinical cancer therapeutics has revolutionized medicine in recent years. DNA sequencing alone may miss clinically actionable variants or identify aberrations that are not being transcribed. In this study we investigated the utility of integrating DNA and RNA sequencing in clinical samples. Methods: A cohort of 32 patient samples were analyzed by WES and RNA sequencing. Differential expression analysis was performed using a cohort of controls. Pathway analysis was performed using Ingenuity Pathway Analysis. WES and RNA analysis detected alterations including SNVs, indels, copy number events, fusions, alternate transcripts, TMB, MSI status and differential expression. Results: A total of 25 CRC (39-78yrs) and 7 pancreatic cancers (PCs) (51-91 yrs) were profiled by WES and RNA seq. A 66 yr old pt with neoplasm of rectosigmoid junction tumor was found to be KRAS wildtype and was treated with cetuximab plus FOLFIRI. Patient failed therapy after 2 yrs and sequencing revealed MET amplification which is a known mechanism of resistance to cetuximab treatment. Further, RNA expression analysis showed 44-fold increase in MET expression along with overexpression of AREG and EREG. Out of the 7 PCs, 3 cases that did not harbor KRAS mutation were found to harbor VTCN1/NRG1 fusion, FGFR1/G3BP2 fusion and BRAF V600E mutation respectively. A 50 year-old stage IV metastatic (met) PC pt was treated with standard of care regimens. Upon relapse the sample was found to harbor VTCN1/NRG1 fusion along with a TERT promoter mutation. RNA expression analysis revealed 54-fold increased expression of NRG1 which may lead to clinical trial enrollment. A 52 year-old male met Stage IV PC, was treated with rucaparib and irinotecan based on prior sequencing data. Upon relapse, the pt went on ATR inhibitor (BAY1895344) and progressed very quickly. Sequencing of the met lesion was found to harbor FGFR1/G3BP2 fusion which was also confirmed by RNA expression. A 55 year-old met pt with PC was treated with standard chemotherapy. Upon disease progression pt was sequenced and based on presence of BRAF V600E, pt was treated with trametinib and dabrafenib. Following disease progression on BRAF/MEKi, met sample was resequenced and RNA expression analysis found increased expression of MET, MACC1 and SMAD7 and 4-fold decrease in PTEN which could potentially cause resistance to BRAF/MEKi therapy. Conclusions: Our study highlights the utility of comprehensive testing integrating genomic and transcriptomic data, in identifying targeted therapy options for cancer patients.
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Lindquist, Michael E., Aaron W. Lifland, Thomas J. Utley, Philip J. Santangelo, and James E. Crowe. "Respiratory Syncytial Virus Induces Host RNA Stress Granules To Facilitate Viral Replication." Journal of Virology 84, no. 23 (September 15, 2010): 12274–84. http://dx.doi.org/10.1128/jvi.00260-10.
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ABSTRACT Mammalian cell cytoplasmic RNA stress granules are induced during various conditions of stress and are strongly associated with regulation of host mRNA translation. Several viruses induce stress granules during the course of infection, but the exact function of these structures during virus replication is not well understood. In this study, we showed that respiratory syncytial virus (RSV) induced host stress granules in epithelial cells during the course of infection. We also showed that stress granules are distinct from cytoplasmic viral inclusion bodies and that the RNA binding protein HuR, normally found in stress granules, also localized to viral inclusion bodies during infection. Interestingly, we demonstrated that infected cells containing stress granules also contained more RSV protein than infected cells that did not form inclusion bodies. To address the role of stress granule formation in RSV infection, we generated a stable epithelial cell line with reduced expression of the Ras-GAP SH3 domain-binding protein (G3BP) that displayed an inhibited stress granule response. Surprisingly, RSV replication was impaired in these cells compared to its replication in cells with intact G3BP expression. In contrast, knockdown of HuR by RNA interference did not affect stress granule formation or RSV replication. Finally, using RNA probes specific for RSV genomic RNA, we found that viral RNA predominantly localized to viral inclusion bodies but a small percentage also interacted with stress granules during infection. These results suggest that RSV induces a host stress granule response and preferentially replicates in host cells that have committed to a stress response.
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Jovanovic, Bogdan, Lisa Schubert, Fabian Poetz, and Georg Stoecklin. "Tagging of RPS9 as a tool for ribosome purification and identification of ribosome-associated proteins." Archives of Biological Sciences, no. 00 (2020): 57. http://dx.doi.org/10.2298/abs20120557j.
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Ribosomes, the catalytic machinery required for protein synthesis, are comprised of 4 ribosomal RNAs and about 80 ribosomal proteins in mammals. Ribosomes further interact with numerous associated factors that regulate their biogenesis and function. As mutations of ribosomal proteins and ribosome associated proteins cause many diseases, it is important to develop tools by which ribosomes can be purified efficiently and with high specificity. Here, we designed a method to purify ribosomes from human cell lines by C-terminally tagging human RPS9, a protein of the small ribosomal subunit. The tag consists of a flag peptide and a streptavidin-binding peptide (SBP) separated by the tobacco etch virus (TEV) protease cleavage site. We demonstrate that RPS9-Flag-TEV-SBP (FTS) is efficiently incorporated into the ribosome without interfering with regular protein synthesis. Using HeLa-GFP-G3BP1 cells stably expressing RPS9-FTS or, as a negative control, mCherry-FTS, we show that complete ribosomes as well as numerous ribosome-associated proteins are efficiently and specifically purified following pull-down of RPS9-FTS using streptavidin beads. This tool will be helpful for the characterization of human ribosome heterogeneity, post-translational modifications of ribosomal proteins, and changes in ribosome-associated factors after exposing human cells to different stimuli and conditions.
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Tsai, Kuen-Nan, Chin-Liew Chong, Yu-Chi Chou, Chien-Chiao Huang, Yi-Ling Wang, Shao-Win Wang, Mong-Liang Chen, Chun-Hong Chen, and Chungming Chang. "Doubly Spliced RNA of Hepatitis B Virus Suppresses Viral Transcription via TATA-Binding Protein and Induces Stress Granule Assembly." Journal of Virology 89, no. 22 (September 2, 2015): 11406–19. http://dx.doi.org/10.1128/jvi.00949-15.
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ABSTRACTThe risk of liver cancer in patients infected with the hepatitis B virus (HBV) and their clinical response to interferon alpha therapy vary based on the HBV genotype. The mechanisms underlying these differences in HBV pathogenesis remain unclear. In HepG2 cells transfected with a mutant HBVG2335Aexpression plasmid that does not transcribe the 2.2-kb doubly spliced RNA (2.2DS-RNA) expressed by wild-type HBV genotype A, the level of HBV pregenomic RNA (pgRNA) was higher than that in cells transfected with an HBV genotype A expression plasmid. By using cotransfection with HBV genotype D and 2.2DS-RNA expression plasmids, we found that a reduction of pgRNA was observed in the cells even in the presence of small amounts of the 2.2DS-RNA plasmid. Moreover, ectopic expression of 2.2DS-RNA in the HBV-producing cell line 1.3ES2 reduced the expression of pgRNA. Further analysis showed that exogenously transcribed 2.2DS-RNA inhibited a reconstituted transcriptionin vitro. In Huh7 cells ectopically expressing 2.2DS-RNA, RNA immunoprecipitation revealed that 2.2DS-RNA interacted with the TATA-binding protein (TBP) and that nucleotides 432 to 832 of 2.2DS-RNA were required for efficient TBP binding. Immunofluorescence experiments showed that 2.2DS-RNA colocalized with cytoplasmic TBP and the stress granule components, G3BP and poly(A)-binding protein 1 (PABP1), in Huh7 cells. In conclusion, our study reveals that 2.2DS-RNA acts as a repressor of HBV transcription through an interaction with TBP that induces stress granule formation. The expression of 2.2DS-RNA may be one of the viral factors involved in viral replication, which may underlie differences in clinical outcomes of liver disease and responses to interferon alpha therapy between patients infected with different HBV genotypes.IMPORTANCEPatients infected with certain genotypes of HBV have a lower risk of hepatocellular carcinoma and exhibit a more favorable response to antiviral therapy than patients infected with other HBV genotypes. Using cultured human hepatoma cells as a model of HBV infection, we found that the expression of 2.2DS-RNA caused a decrease in HBV replication. In cultured cells, the ectopic expression of 2.2DS-RNA obviously reduced the intracellular levels of HBV mRNAs. Our analysis of the 2.2DS-RNA-mediated suppression of viral RNA expression showed that 2.2DS-RNA inhibited transcription via binding to the TATA-binding protein and stress granule proteins. Our findings suggest that the 2.2DS-RNA acts as a suppressive noncoding RNA that modulates HBV replication, which may in turn influence the development of chronic hepatitis B.
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Lin, Che-Hsuan, Hsun-Hua Lee, Wei-Min Chang, Fei-Peng Lee, Lung-Che Chen, Long-Sheng Lu, and Yuan-Feng Lin. "FOXD1 Repression Potentiates Radiation Effectiveness by Downregulating G3BP2 Expression and Promoting the Activation of TXNIP-Related Pathways in Oral Cancer." Cancers 12, no. 9 (September 21, 2020): 2690. http://dx.doi.org/10.3390/cancers12092690.
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Radiotherapy is commonly used to treat oral cancer patients in the current clinics; however, a subpopulation of patients shows poor radiosensitivity. Therefore, the aim of this study is to identify a biomarker or druggable target to enhance the effectiveness of radiotherapy on oral cancer patients. By performing an in silico analysis against public databases, we found that the upregulation of FOXD1, a gene encoding forkhead box d1 (Foxd1), is extensively detected in primary tumors compared to normal tissues and associated with a poor outcome in oral cancer patients receiving irradiation treatment. Moreover, our data showed that the level of FOXD1 transcript is causally relevant to the effective dosage of irradiation in a panel of oral cancer cell lines. The FOXD1 knockdown (FOXD1-KD) dramatically suppressed the colony-forming ability of oral cancer cells after irradiation treatment. Differentially expressed genes analysis showed that G3BP2, a negative regulator of p53, is predominantly repressed after FOXD1-KD and transcriptionally regulated by Foxd1, as judged by a luciferase-based promoter assay in oral cancer cells. Gene set enrichment analysis significantly predicted the inhibition of E2F-related signaling pathway but the activation of the interferons (IFNs) and p53-associated cellular functions, which were further validated by luciferase reporter assays in the FOXD1-KD oral cancer cells. Robustly, our data showed that FOXD1-KD fosters the expression of TXNIP, a downstream effector of IFN signaling and activator of p53, in oral cancer cells. These findings suggest that FOXD1 targeting might potentiate the anti-cancer effectiveness of radiotherapy and promote immune surveillance on oral cancer.
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Hosono, Naoko, Hideki Makishima, Bartlomiej P. Przychodzen, Thomas LaFramboise, Chantana Polprasert, Michael J. Clemente, Jacqueline Boultwood, Mikkael A. Sekeres, and Jaroslaw P. Maciejewski. "Analysis of Clonal Hierarchy Shows That Other Ancestral Events May Precede Evolution of Del(5q) in Myeloid Neoplasms." Blood 124, no. 21 (December 6, 2014): 4605. http://dx.doi.org/10.1182/blood.v124.21.4605.4605.
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Abstract The molecular pathogenesis of myeloid neoplasms characterized by 5q deletion (del(5q)) has not been completely elucidated. While some pathomorphologic features including e.g., megakaryocytic and erythroid dysplasia, have been associated with specific genes within minimal common deleted regions (CDR), genes responsible for clonal advantage and expansion have not been identified. It is not clear if haploinsufficiency of one or multiple genes within del(5q) is responsible for clonal evolution or whether mutations in those genes or other genes located in other genomic areas are present. Moreover, with the recognition of intra-tumor diversity and hierarchical clonal architecture, it may be possible to establish whether del(5q) or other lesions, including common somatic mutations, constitute the ancestral event in the pathophysiologic cascade. We performed a comprehensive mutational screen in 124 patients with del(5q), including 59 patients studied by whole exome sequencing (WES) and 65 by targeted deep NGS of genes within the deleted area and the other most commonly mutated genes as previously determined in WES cohorts. To identify pathogenic genes, those most consistently found to be haploinsufficient in del(5q) were matched for the presence of mutations in diploid cases. For the purpose of this study haploinsufficiency was quantitated based on the number of cases with del(5q) showing <60% expression of the corresponding genes. E.g.,HDAC3 in 81%, PPP2CA in 62% and RPS14 in 14% of cases with del(5q). For all somatic mutations, we also describe the clonal composition based on deep sequencing in serial samples and analyses of variant allelic frequency. Finally, we compare the clonal size for individual mutations with that of del(5q). The latter was accomplished by calculation of clonal size based on allelic imbalance for informative SNPs present within deleted regions in heterozygous configurations in germ line samples. The average deviation from the ideal 50/50 distribution in tumor samples allowed for precise calculation of the proportion of cells in the sample affected by the deletion. Using this approach, there was a good correlation to the size of del(5q) clone by FISH (r=.94) Our results demonstrate that 10/14 genes were haploinsufficient within the CDR, but only 2 hemizygous somatic mutations were identified. However, 12 mutations in 7 genes (MATR3, SH3TC2, CSNK1A1, PDGFRB, CD74, FAT2 and G3BP1) were present with the area corresponding to the CDR in diploid cases. TP53 mutations were more commonly associated with del(5q) (73%, vs. 27% in diploid 5, p<.001) and were particularly frequent in patients affected with 2 commonly retained regions (CRR1;5q11.1-5q14.2 and CRR2; 5q34-qter), where they were found in 81% of cases (30/37) vs. 19% (7/30) among CDR deletions (p<.001). In lower-risk MDS, mutations were detected in 11% of deletion cases, whereas they were only found in 5% of diploid chr5 (p<.0001). In higher-risk MDS, TP53 mutation were found in 42% of del(5q) vs. 4% of diploid chr5 (p<.0001). Similarly, 45% patients with concomitant -7/del(7q) and del(5q) had TP53 mutations. The most common mutation associated with del(5q) was TP53, while mutations of FLT3, NRAS or TET2 were significantly mutually exclusive (p=0.03, 0.04 and 0.03; respectively). Next we determined the earliest somatic event by comparing of clonal size of the associated lesions. Del(5q) was present in 17-98% of tumor cells. We identified three theoretical possibilities as to the clonal architecture of del(5q) myeloid neoplasms: i) Tumors in which driver somatic mutations precede del(5q) (35%), ii) those in which del(5q) appears to precede any other somatic mutation (6%) and iii) the succession cannot be determined because of very expanded clones of similar size (“clonal saturation”) i.e., these cases were not informative. For cases in which del(5q) was a secondary lesion, TP53 was the ancestral event 64% of the time, and DNMT3A 27% of the time. The TP53 mutation was detected as a secondary event in 1 of 2 samples in which del(5q) was found to be ancestral. In sum, our results suggest that del(5q) is not universally an ancestral event. The TP53 mutation is the most common mutation in del(5q) and may also serve as ancestral event. While UPD17p and hemizygocity for TP53 can be found in 33% of TP53 mutant cases, most of the detected TP53 mutations were likely to heterozygous, and therefore the clonal size was not overestimated. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen Corp: Membership on an entity's Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity's Board of Directors or advisory committees.
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Boultwood, Jacqueline, Carrie Fidler, Amanda J. Strickson, Fiona Watkins, Susana Gama, Lyndal Kearney, Sabrina Tosi, et al. "Narrowing and genomic annotation of the commonly deleted region of the 5q− syndrome." Blood 99, no. 12 (June 15, 2002): 4638–41. http://dx.doi.org/10.1182/blood.v99.12.4638.
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The 5q− syndrome is the most distinct of the myelodysplastic syndromes, and the molecular basis for this disorder remains unknown. We describe the narrowing of the common deleted region (CDR) of the 5q− syndrome to the approximately 1.5-megabases interval at 5q32 flanked by D5S413 and theGLRA1 gene. The Ensembl gene prediction program has been used for the complete genomic annotation of the CDR. The CDR is gene rich and contains 24 known genes and 16 novel (predicted) genes. Of 40 genes in the CDR, 33 are expressed in CD34+ cells and, therefore, represent candidate genes since they are expressed within the hematopoietic stem/progenitor cell compartment. A number of the genes assigned to the CDR represent good candidates for the 5q− syndrome, including MEGF1, G3BP, and several of the novel gene predictions. These data now afford a comprehensive mutational/expression analysis of all candidate genes assigned to the CDR.
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Huang, Yanping, Roman P. Wernyj, Darrell D. Norton, Patricia Precht, Maria-Cristina Seminario, and Ronald L. Wange. "Modulation of specific protein expression levels by PTEN: identification of AKAP121, DHFR, G3BP, Rap1 and RCC1 as potential targets of PTEN." Oncogene 24, no. 23 (March 21, 2005): 3819–29. http://dx.doi.org/10.1038/sj.onc.1208527.
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Martin, Sophie, Nicolas Bellora, Juan González-Vallinas, Manuel Irimia, Karim Chebli, Marion de Toledo, Monika Raabe, et al. "Preferential binding of a stable G3BP ribonucleoprotein complex to intron-retaining transcripts in mouse brain and modulation of their expression in the cerebellum." Journal of Neurochemistry 139, no. 3 (October 18, 2016): 349–68. http://dx.doi.org/10.1111/jnc.13768.
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Neumann, Frank, Daniela C. Bruennert, Anne-Marie Koch, Ingmar Bruns, Norbert Gattermann, Ralf Kronenwett, and Rainer Haas. "Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562." Blood 110, no. 11 (November 16, 2007): 4540. http://dx.doi.org/10.1182/blood.v110.11.4540.4540.
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Abstract Introduction: Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Particular emphasis was put on 1539 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in first chronic phase in comparison to CD34+ cells from normal bone marrow (Diaz-Blanco et al., Leukemia 2006). Methods: Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5x107 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration, IC 50). FISH analysis confirmed the K562 cell line to be BCR-ABL positive. Gene expression data of the treated cells were compared with the data of untreated cells. In addition, proliferation (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. A colony assay was performed to see differences in cell growth. Results: Looking at those 1539 differentially expressed genes in K562 cells which distinguish patients with CML from healthy donors, we found that Imatinib led to a significant downregulation of 187 and upregulation of 45 genes. In general, Nilotinib had a more pronounced effect than Imatinib regarding the number of genes affected and the degree of suppression. It caused downregulation of 418 and upregulation of 41 genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib such as those related to bcr-abl signalling (Lyn, BCL2, Myc, PIK3CB, G3BP2). Downregulation of genes involved in cell cycle (CDK2, ORC5L, MCM3, POLE2, CCNG1) was only observed following Nilotinib exposure. The stronger effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5 fold greater following treatment with Nilotinib in comparison to Imatinib after 24 hours. Treatment with either Imatinib or Nilotinib produced a similar apoptotic rate and similar decrease in cell numbers after 96 hours. In the colony forming assay, the controls (K562 cells incubated with DMSO only) displayed strong leukemic growth which was inhibited by both Nilotinib and Imatinib, allowing only small clusters to appear. Conclusion: Nilotinib is apparently more potent than Imatinib with regard to the number of genes downregulated and the degree of their suppression. Many of the suppressed genes are associated with bcr-abl signalling and cell cycle.