Journal articles on the topic 'Functional mRNA'
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1
Yu, Jia, and J. Eric Russell. "Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA." Molecular and Cellular Biology 21, no. 17 (September 1, 2001): 5879–88. http://dx.doi.org/10.1128/mcb.21.17.5879-5888.2001.
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ABSTRACT Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.
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Alekhina, Olga, Ilya Terenin, Sergey Dmitriev, and Konstantin Vassilenko. "Functional Cyclization of Eukaryotic mRNAs." International Journal of Molecular Sciences 21, no. 5 (February 29, 2020): 1677. http://dx.doi.org/10.3390/ijms21051677.
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The closed-loop model of eukaryotic translation states that mRNA is circularized by a chain of the cap-eIF4E-eIF4G-poly(A)-binding protein (PABP)-poly(A) interactions that brings 5′ and 3′ ends together. This circularization is thought to promote the engagement of terminating ribosomes to a new round of translation at the same mRNA molecule, thus enhancing protein synthesis. Despite the general acceptance and the elegance of the hypothesis, it has never been proved experimentally. Using continuous in situ monitoring of luciferase synthesis in a mammalian in vitro system, we show here that the rate of translation initiation at capped and polyadenylated reporter mRNAs increases after the time required for the first ribosomes to complete mRNA translation. Such acceleration strictly requires the presence of a poly(A)-tail and is abrogated by the addition of poly(A) RNA fragments or m7GpppG cap analog to the translation reaction. The optimal functional interaction of mRNA termini requires 5′ untranslated region (UTR) and 3′ UTR of moderate lengths and provides stronger acceleration, thus a longer poly(A)-tail. Besides, we revealed that the inhibitory effect of the dominant negative R362Q mutant of initiation factor eIF4A diminishes in the course of translation reaction, suggesting a relaxed requirement for ATP. Taken together, our results imply that, upon the functional looping of an mRNA, the recycled ribosomes can be recruited to the start codon of the same mRNA molecule in an eIF4A-independent fashion. This non-canonical closed-loop assisted reinitiation (CLAR) mode provides efficient translation of the functionally circularized mRNAs.
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Yang, Edward, Erik van Nimwegen, Mihaela Zavolan, Nikolaus Rajewsky, Mark Schroeder, Marcelo Magnasco, and James E. Darnell. "Decay Rates of Human mRNAs: Correlation With Functional Characteristics and Sequence Attributes." Genome Research 13, no. 8 (August 2003): 1863–72. http://dx.doi.org/10.1101/gr.1272403.
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Although mRNA decay rates are a key determinant of the steady-state concentration for any given mRNA species, relatively little is known, on a population level, about what factors influence turnover rates and how these rates are integrated into cellular decisions. We decided to measure mRNA decay rates in two human cell lines with high-density oligonucleotide arrays that enable the measurement of decay rates simultaneously for thousands of mRNA species. Using existing annotation and the Gene Ontology hierarchy of biological processes, we assign mRNAs to functional classes at various levels of resolution and compare the decay rate statistics between these classes. The results show statistically significant organizational principles in the variation of decay rates among functional classes. In particular, transcription factor mRNAs have increased average decay rates compared with other transcripts and are enriched in “fast-decaying” mRNAs with half-lives <2 h. In contrast, we find that mRNAs for biosynthetic proteins have decreased average decay rates and are deficient in fast-decaying mRNAs. Our analysis of data from a previously published study of Saccharomyces cerevisiae mRNA decay shows the same functional organization of decay rates, implying that it is a general organizational scheme for eukaryotes. Additionally, we investigated the dependence of decay rates on sequence composition, that is, the presence or absence of short mRNA motifs in various regions of the mRNA transcript. Our analysis recovers the positive correlation of mRNA decay with known AU-rich mRNA motifs, but we also uncover further short mRNA motifs that show statistically significant correlation with decay. However, we also note that none of these motifs are strong predictors of mRNA decay rate, indicating that the regulation of mRNA decay is more complex and may involve the cooperative binding of several RNA-binding proteins at different sites.
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Mauger, David M., B. Joseph Cabral, Vladimir Presnyak, Stephen V. Su, David W. Reid, Brooke Goodman, Kristian Link, et al. "mRNA structure regulates protein expression through changes in functional half-life." Proceedings of the National Academy of Sciences 116, no. 48 (November 11, 2019): 24075–83. http://dx.doi.org/10.1073/pnas.1908052116.
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Messenger RNAs (mRNAs) encode information in both their primary sequence and their higher order structure. The independent contributions of factors like codon usage and secondary structure to regulating protein expression are difficult to establish as they are often highly correlated in endogenous sequences. Here, we used 2 approaches, global inclusion of modified nucleotides and rational sequence design of exogenously delivered constructs, to understand the role of mRNA secondary structure independent from codon usage. Unexpectedly, highly expressed mRNAs contained a highly structured coding sequence (CDS). Modified nucleotides that stabilize mRNA secondary structure enabled high expression across a wide variety of primary sequences. Using a set of eGFP mRNAs with independently altered codon usage and CDS structure, we find that the structure of the CDS regulates protein expression through changes in functional mRNA half-life (i.e., mRNA being actively translated). This work highlights an underappreciated role of mRNA secondary structure in the regulation of mRNA stability.
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Pilkington, Guy R., and Roy Parker. "Pat1 Contains Distinct Functional Domains That Promote P-Body Assembly and Activation of Decapping." Molecular and Cellular Biology 28, no. 4 (December 17, 2007): 1298–312. http://dx.doi.org/10.1128/mcb.00936-07.
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ABSTRACT The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P bodies. The Pat1 protein is a key component of this complex and an important activator of decapping, yet little is known about its function. In this work, we analyze Pat1 in Saccharomyces cerevisiae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1: one promoting translation repression and P-body assembly and a second domain promoting mRNA decapping after assembly of the mRNA into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.
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Chang, Jeong Ho, and Liang Tong. "Structural and functional studies of the decapping exoribonucleases." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1397. http://dx.doi.org/10.1107/s2053273314086021.
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Recent studies showed that two homologous yeast proteins, Rai1 and Dxo1, function in a quality control mechanism to clear cells of incompletely 5' end-capped messenger RNAs (mRNAs). Rai1 possesses a novel decapping activity that can remove the entire cap structure dinucleotide from an mRNA. This activity is targeted preferentially towards mRNAs with unmethylated caps in contrast to the canonical decapping enzyme, Dcp2, which targets mRNAs with a methylated cap. Dxo1 also has robust decapping activity on RNAs with unmethylated caps, but it has no detectable pyrophosphohydrolase activity. Unexpectedly, we found that Dxo1 also possesses distributive, 5'-3' exoribonuclease activity, and we named Dxo1 (originally Ydr370C) for this new eukaryotic enzyme with both decapping and exonuclease activities. Studies of yeast in which both Dxo1 and Rai1 are disrupted reveal that mRNAs with incomplete caps are produced even under normal growth conditions, in sharp contrast to current understanding of the capping process. Here, we introduce that their mammalian homolog, Dom3Z (referred to as DXO), possesses pyrophosphohydrolase, decapping, and 5'-3' exoribonuclease activities. Surprisingly, we found that DXO preferentially degrades defectively capped pre-mRNAs in cells. Additional studies show that incompletely capped pre-mRNAs are inefficiently spliced at all introns, a fact that contrasts with current understanding, and are also poorly cleaved for polyadenylation. Crystal structures of DXO in complex with substrate mimic and products at a resolution of up to 1.5 Å provide elegant insights into the catalytic mechanism and molecular basis for their three apparently distinct activities. Our data reveal a pre-mRNA 5' end capping quality control mechanism in mammalian cells, indicating DXO as the central player for this mechanism, and demonstrate an unexpected intimate link between proper 5' end capping and subsequent pre-mRNA processing.
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Green-Willms, Noelle S., Thomas D. Fox, and Maria C. Costanzo. "Functional Interactions between Yeast Mitochondrial Ribosomes and mRNA 5′ Untranslated Leaders." Molecular and Cellular Biology 18, no. 4 (April 1, 1998): 1826–34. http://dx.doi.org/10.1128/mcb.18.4.1826.
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ABSTRACT Translation of mitochondrial mRNAs in Saccharomyces cerevisiae depends on mRNA-specific translational activators that recognize the 5′ untranslated leaders (5′-UTLs) of their target mRNAs. We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5′-UTLs of both the COX2 and COX3 mRNAs, indicating a general function in translational activation. One gene, MRP21, encodes a protein with a domain related to the bacterial ribosomal protein S21 and to unidentified proteins of several animals. The other gene, MRP51, encodes a novel protein whose only known homolog is encoded by an unidentified gene in S. kluyveri. Deletion of either MRP21 or MRP51 completely blocked mitochondrial gene expression. Submitochondrial fractionation showed that both Mrp21p and Mrp51p cosediment with the mitochondrial ribosomal small subunit. The suppressor mutations are missense substitutions, and those affecting Mrp21p alter the region homologous to E. coli S21, which is known to interact with mRNAs. Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not generally increase translational efficiency, since some alleles that strongly suppress 5′-UTL mutations fail to suppress initiation codon mutations. We propose that mitochondrial ribosomes themselves recognize a common feature of mRNA 5′-UTLs which, in conjunction with mRNA-specific translational activation, is required for organellar translation initiation.
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Yang, Feng, Yong Peng, Elizabeth L. Murray, Yuichi Otsuka, Nancy Kedersha, and Daniel R. Schoenberg. "Polysome-Bound Endonuclease PMR1 Is Targeted to Stress Granules via Stress-Specific Binding to TIA-1." Molecular and Cellular Biology 26, no. 23 (September 18, 2006): 8803–13. http://dx.doi.org/10.1128/mcb.00090-06.
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ABSTRACT The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the α subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional ∼680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs.
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GRÖLZ, Daniel, and Michael BACHMANN. "The nuclear autoantigen La/SS-associated antigen B: one gene, three functional mRNAs." Biochemical Journal 323, no. 1 (April 1, 1997): 151–58. http://dx.doi.org/10.1042/bj3230151.
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Transcription of the gene encoding for the nuclear autoantigen La resulted in three mRNA forms. A promoter switching combined with an alternative splicing pathway replaced exon 1 with either exon 1´ or exon 1´´. The exon 1´´ donor splice site was located 4 nts downstream of the exon 1´ donor splice site. All three La mRNA forms were expressed in all the tissues analysed including peripheral blood lymphocytes, liver, fetal spleen, cultured primary endothelial cells, and mouse LTA cell lines permanently transfected with the human La gene. Both the exons 1´ and 1´´ had unusual structures. They contained GC-rich regions and an oligo(U)-tail of 23 uridine residues. Moreover, they encoded for three open reading frames upstream of the La protein reading frame. In spite of this unusual structure, when exon 1´ or exon 1´´ La mRNAs were expressed in transfected mouse LTA cells, both La mRNAs were translated to nuclear La protein, indicating that all La mRNA forms are functional mRNAs.
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Hennigan, A. N., and A. Jacobson. "Functional mapping of the translation-dependent instability element of yeast MATalpha1 mRNA." Molecular and Cellular Biology 16, no. 7 (July 1996): 3833–43. http://dx.doi.org/10.1128/mcb.16.7.3833.
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The determinants of mRNA stability include specific cis-acting destabilizing sequences located within mRNA coding and noncoding regions. We have developed an approach for mapping coding-region instability sequences in unstable yeast mRNAs that exploits the link between mRNA translation and turnover and the dependence of nonsense-mediated mRNA decay on the activity of the UPF1 gene product. This approach, which involves the systematic insertion of in-frame translational termination codons into the coding sequence of a gene of interest in a upf1delta strain, differs significantly from conventional methods for mapping cis-acting elements in that it causes minimal perturbations to overall mRNA structure. Using the previously characterized MATalpha1 mRNA as a model, we have accurately localized its 65-nucleotide instability element (IE) within the protein coding region. Termination of translation 5' to this element stabilized the MATalpha1 mRNA two- to threefold relative to wild-type transcripts. Translation through the element was sufficient to restore an unstable decay phenotype, while internal termination resulted in different extents of mRNA stabilization dependent on the precise location of ribosome stalling. Detailed mutagenesis of the element's rare-codon/AU-rich sequence boundary revealed that the destabilizing activity of the MATalpha1 IE is observed when the terminal codon of the element's rare-codon interval is translated. This region of stability transition corresponds precisely to a MATalpha1 IE sequence previously shown to be complementary to 18S rRNA. Deletion of three nucleotides 3' to this sequence shifted the stability boundary one codon 5' to its wild-type location. Conversely, constructs containing an additional three nucleotides at this same location shifted the transition downstream by an equivalent sequence distance. Our results suggest a model in which the triggering of MATalpha1 mRNA destabilization results from establishment of an interaction between translating ribosomes and a downstream sequence element. Furthermore, our data provide direct molecular evidence for a relationship between mRNA turnover and mRNA translation.
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Wang, Y., C. L. Liu, J. D. Storey, R. J. Tibshirani, D. Herschlag, and P. O. Brown. "Precision and functional specificity in mRNA decay." Proceedings of the National Academy of Sciences 99, no. 9 (April 23, 2002): 5860–65. http://dx.doi.org/10.1073/pnas.092538799.
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LIU, S. W. "Functional analysis of mRNA scavenger decapping enzymes." RNA 10, no. 9 (September 1, 2004): 1412–22. http://dx.doi.org/10.1261/rna.7660804.
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Pieper, Dorothea, Susann Schirmer, Alexander T. Prechtel, Ralph H. Kehlenbach, Joachim Hauber, and Jan Chemnitz. "Functional Characterization of the HuR:CD83 mRNA Interaction." PLoS ONE 6, no. 8 (August 4, 2011): e23290. http://dx.doi.org/10.1371/journal.pone.0023290.
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MacNicol, Melanie, Chad Cragle, Karthik Arumugam, Bruno Fosso, Graziano Pesole, and Angus MacNicol. "Functional Integration of mRNA Translational Control Programs." Biomolecules 5, no. 3 (July 21, 2015): 1580–99. http://dx.doi.org/10.3390/biom5031580.
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Gunnery, S., and M. B. Mathews. "Functional mRNA can be generated by RNA polymerase III." Molecular and Cellular Biology 15, no. 7 (July 1995): 3597–607. http://dx.doi.org/10.1128/mcb.15.7.3597.
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Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.
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Bauer, Rebekka, Sofie Patrizia Meyer, Karolina Anna Kloss, Vanesa Maria Guerrero Ruiz, Samira Reuscher, You Zhou, Dominik Christian Fuhrmann, Kathi Zarnack, Tobias Schmid, and Bernhard Brüne. "Functional RNA Dynamics Are Progressively Governed by RNA Destabilization during the Adaptation to Chronic Hypoxia." International Journal of Molecular Sciences 23, no. 10 (May 22, 2022): 5824. http://dx.doi.org/10.3390/ijms23105824.
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Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
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Wiederhold, Katrin, and Lori A. Passmore. "Cytoplasmic deadenylation: regulation of mRNA fate." Biochemical Society Transactions 38, no. 6 (November 24, 2010): 1531–36. http://dx.doi.org/10.1042/bst0381531.
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The poly(A) tail of mRNA has an important influence on the dynamics of gene expression. On one hand, it promotes enhanced mRNA stability to allow production of the protein, even after inactivation of transcription. On the other hand, shortening of the poly(A) tail (deadenylation) slows down translation of the mRNA, or prevents it entirely, by inducing mRNA decay. Thus deadenylation plays a crucial role in the post-transcriptional regulation of gene expression, deciding the fate of individual mRNAs. It acts both in basal mRNA turnover, as well as in temporally and spatially regulated translation and decay of specific mRNAs. In the present paper, we discuss mRNA deadenylation in eukaryotes, focusing on the main deadenylase, the Ccr4–Not complex, including its composition, regulation and functional roles.
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Winzen, Reinhard, Basant Kumar Thakur, Oliver Dittrich-Breiholz, Meera Shah, Natalie Redich, Sonam Dhamija, Michael Kracht, and Helmut Holtmann. "Functional Analysis of KSRP Interaction with the AU-Rich Element of Interleukin-8 and Identification of Inflammatory mRNA Targets." Molecular and Cellular Biology 27, no. 23 (October 1, 2007): 8388–400. http://dx.doi.org/10.1128/mcb.01493-07.
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ABSTRACT mRNA stability is a major determinant of inflammatory gene expression. Rapid degradation of interleukin-8 (IL-8) mRNA is imposed by a bipartite AU-rich element (ARE) in the 3′ untranslated region (R. Winzen et al., Mol. Cell. Biol. 24:4835-4847, 2004). Small interfering RNA-mediated knockdown of the ARE-binding protein KSRP resulted in stabilization of IL-8 mRNA or of a β-globin reporter mRNA containing the IL-8 ARE. Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.
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Herrera, V. L., J. R. Emanuel, N. Ruiz-Opazo, R. Levenson, and B. Nadal-Ginard. "Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications." Journal of Cell Biology 105, no. 4 (October 1, 1987): 1855–65. http://dx.doi.org/10.1083/jcb.105.4.1855.
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We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.
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van Zalen, Sebastiaan, and J. Eric Russell. "Two Novel Trans-Acting Factors Dictate the High Cytoplasmic Stability of β-Globin mRNA." Blood 116, no. 21 (November 19, 2010): 644. http://dx.doi.org/10.1182/blood.v116.21.644.644.
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Abstract Abstract 644 The efficient accumulation of hemoglobin in mature erythrocytes is critically dependent upon the high stabilities of mRNAs encoding human α- and β-globin proteins. These mRNAs are likely to be stabilized by interactions between one or more trans-acting regulatory factors that target defined cis-acting elements within their 3′UTRs. Several ubiquitous factors that are known to bind to the β-globin 3′UTR (including αCP, PTBP1, and nucleolin) are largely restricted to the nucleus and therefore unlikely to contribute to regulatory processes affecting β-globin mRNA in the cytoplasm. Consequently, we conducted a series of experiments that identify and characterize mRNA-binding factors that dictate the properties of β-globin mRNA in the cytoplasm of erythroid progenitor cells. Using electrophoretic gel mobility shift analyses (EMSA), we defined a characteristic mRNP complex that assembles on the β-globin 3′UTR in cytoplasmic extract–but not nuclear extract–prepared from erythroid K562 cells. This mRNP ‘β-complex’ appears to be erythroid-specific, as it fails to assemble in extracts prepared from non-erythroid HeLa or HEK cells. The 3′UTR binding site for the β-complex was identified using an EMSA-competition approach; remarkably, the target sequence is encompassed within a 12-nt region previously identified as a functional determinant of β-globin mRNA stability in in vivo analyses. Additional experiments fine-mapped the β-complex binding site to a GGGGG pentanucleotide motif within the mRNA-stabilizing region. The functional importance of the pentanucleotide was illustrated by mRNA decay experiments in intact erythroid K562 cells showing that full-length β-globin mRNAs are destabilized by introduction of the same GGGGG->CCGGG mutation that ablates β-complex assembly in EMSA analyses. To identify trans-factors that comprise the β-complex, we performed affinity chromatography using ssDNA probes corresponding to the β-complex binding motif. The native 3′UTR probe retained 42- and 47-kDa proteins, while a probe carrying the CCGGG mutation failed to bind either factor. Subsequent LC/MS/MS analyses identified the two proteins as YB-1 and AUF-1. The identities of these two mRNA-binding factors, which have previously been implicated in the post-transcriptional regulation of heterologous mRNAs, were subsequently confirmed by immunoblot of the protein-DNA complexes. Subsequent analyses suggested a functional role for both factors: EMSA supershift experiments confirmed that YB-1 is a component of the β-complex, and RNA immunoprecipitation analyses demonstrated that both YB-1 and AUF-1 specifically bind to β-globin mRNA in vivo in intact erythroid K562 cells. Collectively, these data identify two novel trans-acting factors that bind to cytoplasmic β-globin mRNA in an erythroid-specific fashion, at a site that dictates its stability in intact cells. We are currently engaged in siRNA knock-down experiments to validate experiments that suggest the importance of these trans-acting factors to the constitutive cytoplasmic stability of β-globin mRNA, as well as structural analyses intended to define RNA-protein and protein-protein interactions that are critical to normal functioning of the β-complex. The results of these experiments have obvious implications for the design of novel therapies for patients with congenital disorders of β-globin gene expression, including sickle cell disease and β thalassemia. Disclosures: No relevant conflicts of interest to declare.
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Chen, C. Y., and A. B. Shyu. "Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements." Molecular and Cellular Biology 14, no. 12 (December 1994): 8471–82. http://dx.doi.org/10.1128/mcb.14.12.8471-8482.1994.
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The metabolic lifetime of mRNA can be specified by specific cis-acting elements within mRNA. One type of element is an adenylate- and uridylate-rich element (ARE) found in the 3' untranslated region of many highly unstable mRNAs for mammalian early-response genes (ERGs). Among the better-characterized members of the ERG family are certain genes encoding nuclear transcription factors. Of particular significance was the finding that their mRNAs decay rapidly with kinetics similar to those of c-fos mRNA. Our previous studies of the c-fos ARE-directed mRNA decay have revealed the existence in this ARE of two structurally distinct and functionally interdependent domains, termed domain I and domain II. We proposed that the c-fos ARE-directed decay is a two-step mechanism in which rapid shortening of the poly(A) tail leads to the decay of the mRNA body and further hypothesized that this is a general mechanism by which the ERG AREs mediate rapid mRNA degradation. To test this hypothesis and to further address the generality of the critical structural characteristics within the c-fos ARE, the RNA-destabilizing functions of more than 10 different AU-rich sequences from various nuclear transcription factor mRNAs have been tested. Consistent with the above-mentioned hypothesis is the observation that mRNAs carrying the functional AREs display a biphasic decay, which is characteristic of the proposed two-step mechanism. Our results indicated that the presence of AUUUA pentanucleotides in an AU-rich region does not always guarantee an RNA-destabilizing function for this region. Our results also led to the identification of a novel class of AU-rich destabilizing elements which contains no AUUUA pentanucleotide. The results of sequence comparison and functional tests revealed that a continuous U-rich sequence is a unique feature among the functional AREs. Finally, our experiments further showed that the c-fos ARE domain II has an RNA decay-enhancing ability upon its fusion to heterologous AU-rich regions and defined for the first time an RNA decay-enhancing element, which we termed the RDE element.
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Chen, C. Y., and A. B. Shyu. "Selective degradation of early-response-gene mRNAs: functional analyses of sequence features of the AU-rich elements." Molecular and Cellular Biology 14, no. 12 (December 1994): 8471–82. http://dx.doi.org/10.1128/mcb.14.12.8471.
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The metabolic lifetime of mRNA can be specified by specific cis-acting elements within mRNA. One type of element is an adenylate- and uridylate-rich element (ARE) found in the 3' untranslated region of many highly unstable mRNAs for mammalian early-response genes (ERGs). Among the better-characterized members of the ERG family are certain genes encoding nuclear transcription factors. Of particular significance was the finding that their mRNAs decay rapidly with kinetics similar to those of c-fos mRNA. Our previous studies of the c-fos ARE-directed mRNA decay have revealed the existence in this ARE of two structurally distinct and functionally interdependent domains, termed domain I and domain II. We proposed that the c-fos ARE-directed decay is a two-step mechanism in which rapid shortening of the poly(A) tail leads to the decay of the mRNA body and further hypothesized that this is a general mechanism by which the ERG AREs mediate rapid mRNA degradation. To test this hypothesis and to further address the generality of the critical structural characteristics within the c-fos ARE, the RNA-destabilizing functions of more than 10 different AU-rich sequences from various nuclear transcription factor mRNAs have been tested. Consistent with the above-mentioned hypothesis is the observation that mRNAs carrying the functional AREs display a biphasic decay, which is characteristic of the proposed two-step mechanism. Our results indicated that the presence of AUUUA pentanucleotides in an AU-rich region does not always guarantee an RNA-destabilizing function for this region. Our results also led to the identification of a novel class of AU-rich destabilizing elements which contains no AUUUA pentanucleotide. The results of sequence comparison and functional tests revealed that a continuous U-rich sequence is a unique feature among the functional AREs. Finally, our experiments further showed that the c-fos ARE domain II has an RNA decay-enhancing ability upon its fusion to heterologous AU-rich regions and defined for the first time an RNA decay-enhancing element, which we termed the RDE element.
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Fuentes, Vanessa, Guadalupe Barrera, Joaquín Sánchez, Roberto Hernández, and Imelda López-Villaseñor. "Functional Analysis of Sequence Motifs Involved in the Polyadenylation of Trichomonas vaginalis mRNAs." Eukaryotic Cell 11, no. 6 (March 30, 2012): 725–34. http://dx.doi.org/10.1128/ec.05322-11.
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ABSTRACTSynthesis of functional mRNA in eukaryotes involves processing of precursor transcripts, including the addition of a poly(A) tail at the 3′ end. A multiprotein complex recognizes a polyadenylation signal, generally the hexanucleotide AAUAAA in metazoans, to direct processing of the pre-mRNA. Based on sequence analysis of several cDNAs, we have previously suggested that the UAAA tetranucleotide (which may include the UAA translation stop codon) could be the polyadenylation signal inTrichomonas vaginalis, a parasitic protozoon that causes human trichomoniasis. This proposal is analyzed here with the aid of a transient-expression system of a reporter gene (catflanked byT. vaginalisactin noncoding sequences). When cells were transfected with a plasmid bearing the original 3′ untranslated region (UTR) sequence containing the UAAA motif, the resultingcatmRNA was polyadenylated similarly to the endogenous actin mRNA. Base changes in the UAAA sequence produced alterations to the polyadenylation site of the reporter mRNAs, while nucleotide substitutions at either side of UAAA did not. Furthermore, relocation of the UAAA motif redirected the processing and polyadenylation of the reporter mRNA. In addition, a pre-mRNA cleavage site for polyadenylation was defined. Interaction ofT. vaginalisproteins with the UAAA motif was shown by electrophoretic mobility shift assays. Based on our findings, we provide evidence that inT. vaginalisthe UAAA tetranucleotide has a role equivalent to that of the metazoan consensus AAUAAA polyadenylation signal.
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Banik, Jewel, Katherine Bronson, Gwen Childs, Linda Hardy, Juchan Lim, Melanie MacNicol, and Angus MacNicol. "OR08-2 Functional Association of the Stem Cell Protein Musashi With LSM14B in Control of mRNA Translation." Journal of the Endocrine Society 6, Supplement_1 (November 1, 2022): A452—A453. http://dx.doi.org/10.1210/jendso/bvac150.941.
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Abstract The Musashi RNA-binding protein functions as a gatekeeper of cell maturation and maintains stem cell plasticity by regulating the translation of target mRNAs. The adult anterior pituitary tissue expresses a high level of Musashi and also demonstatres a high level of cell plasticity, indicating a Musashi-dependent function in maintaining endocrine homeostasis in the anterior pituitary. Towards understanding the mechanism(s) by which Musashi functions to control cell plasticity, we have identified co-associated proteins necessary for Musashi function. The two Musashi isoforms (Musashi1 and Musashi2) are conserved across species and are structurally characterized by two N-terminal RNA-recognition motifs (RRMs, that associate with specific sequences in the 3' untranslated region of mRNAs targets) and a disordered C-terminus. Musashi uses these domains to selectively repress, or to activate, mRNA translation in a manner specific to target mRNA and cell context. Both Musashi isoforms contain two C-terminal regulatory serine residues that require phosphorylation to enable mRNA translational activation. However, the Musashi proteins do not have inherent mRNA translation function and utilize co-associated protein complexes to mediate mRNA translation. We have previously identified Musashi1 binding protein partners through mass spectrometry. Here, we have identified the protein interactions that are required for Musashi1-dependent translational activation. Knockdown of target protein levels in Xenopus laevis oocytes indicated that the PABP4, CELF2, LSM14A/B, ELAVL1, ELAVL2, ELAVL4, and PUM1 proteins are required for Musashi target mRNA translational activation. We further determined that the effect of LSM14A/B dual knockdown was in fact specific to LSM14B alone. LSM14B is an RNA-binding protein that is required for RNA granule formation, RNA transcript metabolism, and mRNA translation. Studies in mouse oocytes have shown that LSM14B is required to maintain transcript levels of the Musashi1 target Cyclin B1 (Ccnb1) mRNA prior to mRNA translation. In the adult murine pituitary, LSM14B is ubiquitously expressed in both hormone-producing cells and in stem cells as indicated through single cell RNA sequencing. These findings indicate that LSM14B and Musashi1 may function cooperatively to regulate pituitary hormone production and cell differentiation in response to changes in physiological demand. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.
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Wei, Jiabo, Haihong Zhu, Qijun Zhang, and Qin Zhang. "Prediction of Functional Genes in Primary Varicose Great Saphenous Veins Using the lncRNA-miRNA-mRNA Network." Computational and Mathematical Methods in Medicine 2022 (September 8, 2022): 1–14. http://dx.doi.org/10.1155/2022/4722483.
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Background. Long noncoding RNAs (lncRNAs) have been widely suggested to bind with the microRNA (miRNA) sites and play roles of competing endogenous RNAs (ceRNAs), which can thus affect and regulate target gene and mRNA expression. Such lncRNA-related ceRNAs are identified to exert vital parts in vascular disease. Nonetheless, it remains unknown about how the lncRNA-miRNA-mRNA network functions in the varicose great saphenous veins. Methods. This study acquired the lncRNA and mRNA expression patterns from the GEO database and identifies the differentially expressed mRNAs and lncRNAs by adopting the R software “limma” package. Then, miRcode, miRDB, miRTarbase, and TargetScan were used to establish the miRNA-mRNA pairs and lncRNA-miRNA pairs. In addition, the lncRNA-miRNA-mRNA ceRNA network was constructed by using Cytoscape. Protein-protein interaction, Gene Ontology functional annotations, and Kyoto Encyclopedia of Genes and Genomes enrichment were carried out to examine the candidate hub genes, the functions of genes, and the corresponding pathways. Results. In line with the preset theory, we constructed ceRNA network comprising 12 lncRNAs, 38 miRNAs, and 149 mRNAs. Kyoto Encyclopedia of Genes and Genomes analysis indicated that the PI3K/Akt signaling pathway played a vital part in the development of varicose great saphenous veins. AC114730, AC002127, and AC073342 were significant biomarkers. At the same time, we predicted the potential miRNA, which may exert a significant influence on the varicose great saphenous veins, namely, miR-17-5p, miR-129-5p, miR-1297, miR-20b-5p, and miR-33a-3p. Conclusion. By performing ceRNA network analysis, our study detects new lncRNAs, miRNAs, and mRNAs, which can be applied as underlying biomarkers of varicose great saphenous veins and as therapeutic targets for the treatment of varicose great saphenous veins.
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Hurst, Laurence D., and Nick G. C. Smith. "Molecular evolutionary evidence that H19 mRNA is functional." Trends in Genetics 15, no. 4 (April 1999): 134–35. http://dx.doi.org/10.1016/s0168-9525(99)01696-0.
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Moteki, Shin, and David Price. "Functional Coupling of Capping and Transcription of mRNA." Molecular Cell 10, no. 3 (September 2002): 599–609. http://dx.doi.org/10.1016/s1097-2765(02)00660-3.
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Shuman, S. "Functional domains of vaccinia virus mRNA capping enzyme." Journal of Biological Chemistry 264, no. 16 (June 1989): 9690–95. http://dx.doi.org/10.1016/s0021-9258(18)60585-x.
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Rajyaguru, Purusharth, and Roy Parker. "RGG motif proteins: Modulators of mRNA functional states." Cell Cycle 11, no. 14 (January 15, 2012): 2594–99. http://dx.doi.org/10.4161/cc.20716.
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Ling, Sharon H. M., Rohini Qamra, and Haiwei Song. "Structural and functional insights into eukaryotic mRNA decapping." Wiley Interdisciplinary Reviews: RNA 2, no. 2 (September 2, 2010): 193–208. http://dx.doi.org/10.1002/wrna.44.
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Tahiri-Alaoui, Abdessamad, Daiki Matsuda, Hongtao Xu, Panopoulos Panagiotis, Luke Burman, Luke S. Lambeth, Lawrence Petherbridge, William James, Vincent Mauro, and Venugopal Nair. "The 5′ Leader of the mRNA Encoding the Marek's Disease Virus Serotype 1 pp14 Protein Contains an Intronic Internal Ribosome Entry Site with Allosteric Properties." Journal of Virology 83, no. 24 (September 30, 2009): 12769–78. http://dx.doi.org/10.1128/jvi.01010-09.
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ABSTRACT We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5′ leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5′ leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.
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van Zalen, Sebastiaan, Alyssa A. Lombardi, Grace R. Jeschke, Elizabeth O. Hexner, and J. Eric Russell. "Auf-1 and YB-1 Independently Regulate β-Globin mRNA Stability Through Interaction with Poly(A) Binding Protein." Blood 120, no. 21 (November 16, 2012): 1020. http://dx.doi.org/10.1182/blood.v120.21.1020.1020.
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Abstract Abstract 1020 The normal expression of Hb A in humans requires the high-level stability of α - and β-globin mRNAs in both transcriptionally active and transcriptionally silenced erythroid progenitors. In contrast to α -globin–whose stability is known to be enhanced by an mRNA-protein (mRNP) complex that assembles on a specific pyrimidine-rich track within its 3'UTR–the structure(s) and mechanism(s) that underlie the high stability of human β-globin mRNA remain poorly defined. We recently identified two RNA-binding proteins, AUF-1 and YB-1, that regulate levels of β-globin mRNA in erythroid progenitors by assembling a cytoplasm-restricted mRNP 'β-complex' on its 3'UTR. The function of the β-complex was predicted by in vitro analyses that mapped its binding to a cis-acting determinant of β-globin mRNA stability, and by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF-1 and YB-1 coordinately ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. The biological importance of the β-complex was subsequently confirmed in human hematopoietic stem cells, where shRNA-mediated knock-down of AUF-1 or YB-1 effected lower levels of β-globin mRNA in cells induced to the erythroid lineage, again implicating their participation in post-transcriptional mechanism(s) regulating the stability of β-globin mRNA. To unambiguously link β-complex activity to β-globin mRNA half-life, we conducted formal in vivo mRNA stability analyses in K562 cells using a β-globin mRNA-specific tetracycline-conditional transcriptional chase strategy. A derivative β-globin mRNA carrying a 5-nt substitution that totally disrupts β-complex assembly (βMut mRNA) displayed a lower half-life than wild-type β-globin mRNA (βWT mRNA), confirming the participation of the β-complex in post-transcriptional regulatory processes. Parallel poly(A) tail length analyses indicated a possible mechanism for this activity, revealing that the βMut mRNA had a shortened steady-state poly(A) tail that truncated faster than the poly(A) tail on βWT mRNA, suggesting a functional interaction between the β-complex and poly(A) tail-associated factors. This observation is fully consistent with the known importance of deadenylation to processes regulating the decay of heterologous mRNAs in several other experimental systems. Subsequent studies supported our model for β-complex/poly(A) tail interaction: electrophoretic gel mobility-shift analyses demonstrated that the β-complex readily assembles on polyadenylated β-globin 3'UTRs but not on corresponding deadenylated 3'UTRs, while RNA affinity capture experiments using K562 cytoplasmic extracts demonstrated that a polyadenylated βWT 3'UTR retains poly(A) binding protein (PABP), while a similar β-complex-deficient βMut 3'UTR fails to bind PABP. Ongoing co-immunoprecipation studies are expected to determine whether the β-complex and PABP are tethered by an interval of mRNA or, alternately, interact directly via a protein-protein interaction. Based upon our previous structural and functional analyses indicating that AUF-1 and YB-1 act redundantly to regulate the cytoplasmic level of β-globin mRNA, we are currently investigating the hypothesis that these two factors also display redundant interactions with the poly(A) tail and its trans-acting binding factors. Our initial RNA affinity analyses confirm this expectation, demonstrating that K562 extracts depleted of either AUF-1 or YB-1 (using an shRNA-knock-down strategy) both maintained the ability to assemble a β-complex as well as facilitate PABP binding to a the polyadenylated βWT 3'UTR. We are presently testing AUF-1 and YB-1 for corresponding functional redundancy (i.e., their abilities to independently induce βWT mRNA stability) using in vivo mRNA tethering experiments in which AUF-1 or YB-1 can be structurally modified to promote their independent interaction with the β-complex binding site. Altogether, these experiments demonstrate that the β-complex, through its component mRNA-binding factors AUF-1 and YB-1, effects the high stability of β-globin mRNA by interacting with PABP. A detailed structural and mechanistic description of this process will be invaluable to the design of novel therapeutics for patients with congenital disorders of β-globin gene expression. Disclosures: No relevant conflicts of interest to declare.
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Ryder, Sean P. "Protein-mRNA interactome capture: cartography of the mRNP landscape." F1000Research 5 (November 3, 2016): 2627. http://dx.doi.org/10.12688/f1000research.9404.1.
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RNA-binding proteins play a variety of roles in cellular physiology. Some regulate mRNA processing, mRNA abundance, and translation efficiency. Some fight off invader RNA through small RNA-driven silencing pathways. Others sense foreign sequences in the form of double-stranded RNA and activate the innate immune response. Yet others, for example cytoplasmic aconitase, act as bi-functional proteins, processing metabolites in one conformation and regulating metabolic gene expression in another. Not all are involved in gene regulation. Some play structural roles, for example, connecting the translational machinery to the endoplasmic reticulum outer membrane. Despite their pervasive role and relative importance, it has remained difficult to identify new RNA-binding proteins in a systematic, unbiased way. A recent body of literature from several independent labs has defined robust, easily adaptable protocols for mRNA interactome discovery. In this review, I summarize the methods and review some of the intriguing findings from their application to a wide variety of biological systems.
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Bryll, Alysia R., and Craig L. Peterson. "Functional interaction between the RNA exosome and the sirtuin deacetylase Hst3 maintains transcriptional homeostasis." Genes & Development 36, no. 1-2 (December 16, 2021): 17–22. http://dx.doi.org/10.1101/gad.348923.121.
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Eukaryotic cells maintain an optimal level of mRNAs through unknown mechanisms that balance RNA synthesis and degradation. We found that inactivation of the RNA exosome leads to global reduction of nascent mRNA transcripts, and that this defect is accentuated by loss of deposition of histone variant H2A.Z. We identify the mRNA for the sirtuin deacetylase Hst3 as a key target for the RNA exosome that mediates communication between RNA degradation and transcription machineries. These findings reveal how the RNA exosome and H2A.Z function together to control a deacetylase, ensuring proper levels of transcription in response to changes in RNA degradation.
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Lallena, M. J., C. Martinez, J. Valcarcel, and I. Correas. "Functional association of nuclear protein 4.1 with pre-mRNA splicing factors." Journal of Cell Science 111, no. 14 (July 30, 1998): 1963–71. http://dx.doi.org/10.1242/jcs.111.14.1963.
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Protein 4.1 is a multifunctional polypeptide that links transmembrane proteins with the underlying spectrin/actin cytoskeleton. Recent studies have shown that protein 4.1 is also present in the nucleus, localized in domains enriched in splicing factors. Here we further analyze the relationship between protein 4. 1 and components of the splicing machinery. Using HeLa nuclear extracts capable of supporting the splicing of pre-mRNAs in vitro, we show that anti-4.1 antibodies specifically immunoprecipitate pre-mRNA and splicing intermediates. Immunodepletion of protein 4.1 from HeLa nuclear extracts results in inhibition of their splicing activity, as assayed with two different pre-mRNA substrates. Coprecipitation of protein 4.1 from HeLa nuclear extracts with proteins involved in the processing of pre-mRNA further suggests an association between nuclear protein 4.1 and components of the splicing apparatus. The molecular cloning of a 4.1 cDNA encoding the isoform designated 4.1E has allowed us to show that this protein is targeted to the nucleus, that it associates with the splicing factor U2AF35, and that its overexpression induces the redistribution of the splicing factor SC35. Based on our combined biochemical and localization results, we propose that 4.1 proteins are part of nuclear structures to which splicing factors functionally associate, most likely for storage purposes.
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Zhang, Ke, Guijun Shang, Abhilash Padavannil, Juan Wang, Ramanavelan Sakthivel, Xiang Chen, Min Kim, et al. "Structural–functional interactions of NS1-BP protein with the splicing and mRNA export machineries for viral and host gene expression." Proceedings of the National Academy of Sciences 115, no. 52 (December 11, 2018): E12218—E12227. http://dx.doi.org/10.1073/pnas.1818012115.
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The influenza virulence factor NS1 protein interacts with the cellular NS1-BP protein to promote splicing and nuclear export of the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix protein and is alternatively spliced into the M2 mRNA, which is translated into the M2 ion channel. These proteins have key functions in viral trafficking and budding. To uncover the NS1-BP structural and functional activities in splicing and nuclear export, we performed proteomics analysis of nuclear NS1-BP binding partners and showed its interaction with constituents of the splicing and mRNA export machineries. NS1-BP BTB domains form dimers in the crystal. Full-length NS1-BP is a dimer in solution and forms at least a dimer in cells. Mutations suggest that dimerization is important for splicing. The central BACK domain of NS1-BP interacts directly with splicing factors such as hnRNP K and PTBP1 and with the viral NS1 protein. The BACK domain is also the site for interactions with mRNA export factor Aly/REF and is required for viral M mRNA nuclear export. The crystal structure of the C-terminal Kelch domain shows that it forms a β-propeller fold, which is required for the splicing function of NS1-BP. This domain interacts with the polymerase II C-terminal domain and SART1, which are involved in recruitment of splicing factors and spliceosome assembly, respectively. NS1-BP functions are not only critical for processing a subset of viral mRNAs but also impact levels and nuclear export of a subset of cellular mRNAs encoding factors involved in metastasis and immunity.
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Yue, Yuan, Suiqing Huang, Zixuan Wu, Keke Wang, Huayang Li, Jian Hou, Xiaolin Huang, Li Luo, Quan Liu, and Zhongkai Wu. "Characterization of mRNA Profiles of Exosomes from Diverse Forms of M2 Macrophages." BioMed Research International 2020 (November 21, 2020): 1–13. http://dx.doi.org/10.1155/2020/1585306.
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Exosomes transmit certain amounts of molecules to specific recipient cells for intercellular communication. Among these molecules, messenger RNAs (mRNAs) may be delivered and translated into proteins in the recipient cells, and these mRNAs are thought to be critical mediators of exosomal functions. There are three subtypes of M2 macrophages (M2Ф), M2aФ, M2bФ, and M2cФ, which have different specific functional programs. The aim of the present study was to screen the mRNA profiles in the exosomes of these macrophage subtypes and to analyze the transcriptomic profile features associated with their specific functions. The mRNA contents of the exosomes isolated from the culture supernatants of the M2Ф subtypes were analyzed and compared using the Illumina HiSeq platform. The results indicated that the exosomes contained particular mRNAs from their source cells and were messengers of cellular functions. Bioinformatics analysis suggested that the exosomal mRNAs from M2bФs are enriched in the Toll-like receptor (TLR), tumor necrosis factor (TNF), NOD-like receptor (NLR), and NF-kappa B (NF-κB) signaling pathways. The mRNA profile of exosomes from M2bФ was distinctly different from that of exosomes from M2aФ and M2cФ and was consistent with the M2bФ cytological characteristic of maintaining a high level of proinflammatory cytokine and regulatory factor production. Therefore, the mRNA profiles revealed several characteristics of the exosomes from diverse forms of M2Ф. Further functional investigations based on these results may advance the understanding of the physiological roles of exosome-transferred mRNAs in MФ functions.
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Guo, Xiaojing, Jialei Yang, Baoyun Liang, Tingting Shen, Yan Yan, Siyun Huang, Jinying Zhou, Jiao Huang, Lian Gu, and Li Su. "Identification of Novel LncRNA Biomarkers and Construction of LncRNA-Related Networks in Han Chinese Patients with Ischemic Stroke." Cellular Physiology and Biochemistry 50, no. 6 (2018): 2157–75. http://dx.doi.org/10.1159/000495058.
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Background/Aims: Long non-coding RNAs (lncRNAs) are potential biomarkers of tumors, cardiac disease, and cerebral disease because of their interaction with coding RNAs. This work focused on ischemic stroke (IS) and aimed to identify novel lncRNA biomarkers and construct lncRNA-related networks in IS. Methods: Differentially expressed lncRNAs were identified using Arraystar Human LncRNA Microarray v4.0, and validated with qRT-PCR. A lncRNA–mRNA co-expression network and a lncRNA–miRNA–mRNA regulatory network were constructed. Functional and pathway analyses were then performed. Results: In total, 560 up-regulated and 690 down-regulated differentially expressed lncRNAs were found (P < 0.05, false discovery rate < 0.05, absolute fold change ≥ 2). qRT-PCR results confirmed that lncRNA-ENST00000568297, lncRNA-ENST00000568243, and lncRNA-NR_046084 exhibited significant differential expression between IS and controls (all P < 0.05). Areas under the curves (AUCs) for these lncRNAs were 0.733, 0.743, and 0.690, respectively, and the combined AUC was 0.843. A coding–noncoding co-expression (CNC) network was constructed based on Pearson’s correlation coefficient. A specific lncRNA–miRNA–mRNA regulatory network of ENST00000568297, ENST00000568243, and NR_046084 was also constructed. Functional annotation of the up- and down-regulated mRNAs was performed. Pathway analysis enriched IS-related pathways with mRNAs in the lncRNA–miRNA–mRNA regulatory network. Conclusion: LncRNA and mRNA expression profiles in human peripheral blood were altered after IS. ENST00000568297, ENST00000568243, and NR_046084 were identified as novel potential diagnostic biomarkers of IS. Analysis of the CNC network and lncRNA–miRNA–mRNA regulatory network suggested that lncRNAs may participate in IS pathophysiology by regulating pivotal miRNAs, mRNAs, or IS-related pathways.
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Nelson, Laura E., and Mark A. Sheridan. "Insulin and growth hormone stimulate somatostatin receptor (SSTR) expression by inducing transcription of SSTR mRNAs and by upregulating cell surface SSTRs." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 291, no. 1 (July 2006): R163—R169. http://dx.doi.org/10.1152/ajpregu.00754.2005.
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This study examined the effects of insulin (INS) and growth hormone (GH) on mRNA and functional expression of somatostatin receptors (SSTRs). Rainbow trout liver was used as a model system to evaluate the direct effects of INS and GH on mRNA expression of three SSTR subtypes characterized previously from this species: SSTR1A, SSTR1B, and SSTR2. INS and GH directly stimulated steady-state levels of all SSTR mRNAs in a concentration- and time-dependent manner; however, the pattern of expression was hormone and SSTR subtype specific. INS stimulated SSTR2 expression to a greater extent than SSTR1A or SSTR1B expression, whereas GH stimulated SSTR2 and SSTR1B expression to a similar extent, with SSTR2 and SSTR1B expression being more responsive to GH than SSTR1A. Whether INS- or GH-stimulated SSTR expression resulted from altered rates of transcription and/or changes in mRNA stability also was investigated. Formation of nascent SSTR transcripts in nuclei isolated from rainbow trout hepatocytes was significantly stimulated by INS and GH. Neither INS nor GH, however, affected the stability of SSTR mRNAs. Functional expression of SSTRs was studied in Chinese hamster ovary (CHO-K1) cells stably transfected with SSTR1A or SSTR1B. Surface expression of functional SSTRs was stimulated by INS and GH. These findings indicate that INS and GH stimulate SSTR expression by regulating transcription of SSTR mRNAs and by increasing functional SSTRs on the cell surface, and they suggest that regulation of SSTRs may be important for the coordination of growth, development, and metabolism of vertebrates.
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Navarro, Estanislao, Adrián Mallén, and Miguel Hueso. "Dynamic Variations of 3′UTR Length Reprogram the mRNA Regulatory Landscape." Biomedicines 9, no. 11 (October 28, 2021): 1560. http://dx.doi.org/10.3390/biomedicines9111560.
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This paper concerns 3′-untranslated regions (3′UTRs) of mRNAs, which are non-coding regulatory platforms that control stability, fate and the correct spatiotemporal translation of mRNAs. Many mRNAs have polymorphic 3′UTR regions. Controlling 3′UTR length and sequence facilitates the regulation of the accessibility of functional effectors (RNA binding proteins, miRNAs or other ncRNAs) to 3′UTR functional boxes and motifs and the establishment of different regulatory landscapes for mRNA function. In this context, shortening of 3′UTRs would loosen miRNA or protein-based mechanisms of mRNA degradation, while 3′UTR lengthening would strengthen accessibility to these effectors. Alterations in the mechanisms regulating 3′UTR length would result in widespread deregulation of gene expression that could eventually lead to diseases likely linked to the loss (or acquisition) of specific miRNA binding sites. Here, we will review the mechanisms that control 3′UTR length dynamics and their alterations in human disorders. We will discuss, from a mechanistic point of view centered on the molecular machineries involved, the generation of 3′UTR variability by the use of alternative polyadenylation and cleavage sites, of mutually exclusive terminal alternative exons (exon skipping) as well as by the process of exonization of Alu cassettes to generate new 3′UTRs with differential functional features.
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Mailliot, Justine, Mirella Vivoli-Vega, and Christiane Schaffitzel. "No-nonsense: insights into the functional interplay of nonsense-mediated mRNA decay factors." Biochemical Journal 479, no. 9 (May 12, 2022): 973–93. http://dx.doi.org/10.1042/bcj20210556.
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Nonsense-mediated messenger RNA decay (NMD) represents one of the main surveillance pathways used by eukaryotic cells to control the quality and abundance of mRNAs and to degrade viral RNA. NMD recognises mRNAs with a premature termination codon (PTC) and targets them to decay. Markers for a mRNA with a PTC, and thus NMD, are a long a 3′-untranslated region and the presence of an exon-junction complex (EJC) downstream of the stop codon. Here, we review our structural understanding of mammalian NMD factors and their functional interplay leading to a branched network of different interconnected but specialised mRNA decay pathways. We discuss recent insights into the potential impact of EJC composition on NMD pathway choice. We highlight the coexistence and function of different isoforms of up-frameshift protein 1 (UPF1) with an emphasis of their role at the endoplasmic reticulum and during stress, and the role of the paralogs UPF3B and UPF3A, underscoring that gene regulation by mammalian NMD is tightly controlled and context-dependent being conditional on developmental stage, tissue and cell types.
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Hernandez, Laura L., Sean W. Limesand, Jayne L. Collier, Nelson D. Horseman, and Robert J. Collier. "The bovine mammary gland expresses multiple functional isoforms of serotonin receptors." Journal of Endocrinology 203, no. 1 (August 4, 2009): 123–31. http://dx.doi.org/10.1677/joe-09-0187.
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Recent studies in dairy cows have demonstrated that serotonergic ligands affect milk yield and composition. Correspondingly, serotonin (5-HT) has been demonstrated to be an important local regulator of lactational homeostasis and involution in mouse and human mammary cells. We determined the mRNA expression of bovine 5-HT receptor (HTR) subtypes in bovine mammary tissue (BMT) and used pharmacological agents to evaluate functional activities of 5-HT receptors. The mRNAs for five receptor isoforms (HTR1B, 2A, 2B, 4, and 7) were identified by conventional real-time (RT)-PCR, RT quantitative PCR, and in situ hybridization in BMT. In addition to luminal mammary epithelial cell expression, HTR4 was expressed in myoepithelium, and HTR1B, 2A, and 2B were expressed in small mammary blood vessels. Serotonin suppressed milk protein mRNA expression (α-lactalbumin and β-casein mRNA) in lactogen-treated primary bovine mammary epithelial cell (BMEC) cultures. To probe the functional activities of individual receptors, caspase-3 activity and expression of α-lactalbumin and β-casein were measured. Both SB22489 (1B antagonist) and ritanserin (2A antagonist) increased caspase-3 activity. Expression of α-lactalbumin and β-casein mRNA levels in BMEC were stimulated by low concentrations of SB224289, ritanserin, or pimozide. These results demonstrate that there are multiple 5-HT receptor isoforms in the bovine mammary gland, and point to profound differences between serotonergic systems of the bovine mammary gland and the human and mouse mammary glands. Whereas human and mouse mammary epithelial cells express predominately the protein for the 5-HT7 receptor, cow mammary epithelium expresses multiple receptors that have overlapping, but not identical, functional activities.
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Mattox, William, M. Elaine McGuffin, and Bruce S. Baker. "A Negative Feedback Mechanism Revealed by Functional Analysis of the Alternative Isoforms of the Drosophila Splicing Regulator transformer-2." Genetics 143, no. 1 (May 1, 1996): 303–14. http://dx.doi.org/10.1093/genetics/143.1.303.
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Abstract The Drosophila sex determination gene transfmer-2 (tra-2) is a splicing regulator that affects the sex-specific processing of several distinct pre-mRNAs. While the tra-2 gene itself is known to produce alternative mRNAs that together encode three different TRA-2 protein isoforms, the respective roles of these isoforms in affecting individual pre-mRNA targets has remained unclear. We have generated transgenic fly strains with mutations affecting specific TRA-2 isoforms to investigate their individual roles in regulating the alternative processing of doublesex, exuperantia and tra-2 pre-mRNA. Our results indicate that in somatic tissues two different isoforms function redundantly to direct female differentiation and female-specific doublesex pre-mRNA splicing. In the male germline, where tra-2 has an essential role in spermatogenesis, a single isoform was found to uniquely perform all necessary functions. This isoform appears to regulate its own synthesis during spermatogenesis through a negative feedback mechanism involving intron retention.
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Culjkovic, Biljana, Ivan Topisirovic, Lucy Skrabanek, Melisa Ruiz-Gutierrez, and Katherine L. B. Borden. "eIF4E promotes nuclear export of cyclin D1 mRNAs via an element in the 3′UTR." Journal of Cell Biology 169, no. 2 (April 18, 2005): 245–56. http://dx.doi.org/10.1083/jcb.200501019.
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The eukaryotic translation initiation factor eIF4E is a critical modulator of cellular growth with functions in the nucleus and cytoplasm. In the cytoplasm, recognition of the 5′ m7G cap moiety on all mRNAs is sufficient for their functional interaction with eIF4E. In contrast, we have shown that in the nucleus eIF4E associates and promotes the nuclear export of cyclin D1, but not GAPDH or actin mRNAs. We determined that the basis of this discriminatory interaction is an ∼100-nt sequence in the 3′ untranslated region (UTR) of cyclin D1 mRNA, we refer to as an eIF4E sensitivity element (4E-SE). We found that cyclin D1 mRNA is enriched at eIF4E nuclear bodies, suggesting these are functional sites for organization of specific ribonucleoproteins. The 4E-SE is required for eIF4E to efficiently transform cells, thereby linking recognition of this element to eIF4E mediated oncogenic transformation. Our studies demonstrate previously uncharacterized fundamental differences in eIF4E-mRNA recognition between the nuclear and cytoplasmic compartments and further a novel level of regulation of cellular proliferation.
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Wu, Wei, Lingxiang Wu, Mengyan Zhu, Ziyu Wang, Min Wu, Pengping Li, Yumin Nie, et al. "miRNA Mediated Noise Making of 3′UTR Mutations in Cancer." Genes 9, no. 11 (November 12, 2018): 545. http://dx.doi.org/10.3390/genes9110545.
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Somatic mutations in 3′-untranslated regions (3′UTR) do not alter amino acids and are considered to be silent in cancers. We found that such mutations can promote tumor progression by altering microRNA (miRNA) targeting efficiency and consequently affecting miRNA–mRNA interactions. We identified 67,159 somatic mutations located in the 3′UTRs of messenger RNAs (mRNAs) which can alter miRNA–mRNA interactions (functional somatic mutations, funcMutations), and 69.3% of these funcMutations (the degree of energy change > 12 kcal/mol) were identified to significantly promote loss of miRNA-mRNA binding. By integrating mRNA expression profiles of 21 cancer types, we found that the expression of target genes was positively correlated with the loss of absolute affinity level and negatively correlated with the gain of absolute affinity level. Functional enrichment analysis revealed that genes carrying funcMutations were significantly enriched in the MAPK and WNT signaling pathways, and analysis of regulatory modules identified eighteen miRNA modules involved with similar cellular functions. Our findings elucidate a complex relationship between miRNA, mRNA, and mutations, and suggest that 3′UTR mutations may play an important role in tumor development.
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Barton, P. J., A. J. Harris, and M. E. Buckingham. "Myosin light chain gene expression in developing and denervated fetal muscle in the mouse." Development 107, no. 4 (December 1, 1989): 819–24. http://dx.doi.org/10.1242/dev.107.4.819.
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We have investigated the accumulation of mRNA transcripts of the atrial (or embryonic) myosin light chain MLC1A (MLC1emb), and the two adult fast muscle myosin light chains (MLC1F and MLC3F) during fetal skeletal muscle development in the mouse. In 15-day fetal muscle, MLC1A is the predominant mRNA detectable, by 18 days MLC1F has become the major transcript and MLC3F mRNA is detectable for the first time. By 12 days after birth, MLC1A transcripts are undetectable and MLC1F and MLC3F are similar in abundance. In fetuses treated with beta-bungarotoxin and which therefore develop in the absence of functional nerve, MLC1A and MLC1F undergo normal transitions but MLC3F mRNA accumulation is significantly retarded. This demonstrates that these myosin light chain mRNAs accumulate with differing kinetics, and that MLC3F mRNA accumulation is nerve-dependent during fetal development. The results are discussed in terms of secondary muscle fibre formation, and in relation to the independent regulation of MLC1F and MLC3F mRNAs which are transcribed from the same gene.
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Vende, Patrice, Maria Piron, Nathalie Castagné, and Didier Poncet. "Efficient Translation of Rotavirus mRNA Requires Simultaneous Interaction of NSP3 with the Eukaryotic Translation Initiation Factor eIF4G and the mRNA 3′ End." Journal of Virology 74, no. 15 (August 1, 2000): 7064–71. http://dx.doi.org/10.1128/jvi.74.15.7064-7071.2000.
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ABSTRACT In contrast to the vast majority of cellular proteins, rotavirus proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically binds the 3′-end consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G. Here we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between the cap structure and the 3′ end of rotavirus mRNA was observed in NSP3-expressing cells. The enhancement of viral mRNA translation by NSP3 was also observed in a rabbit reticulocyte lysate translation system supplemented with recombinant NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding domains are both required to enhance the translation of viral mRNA. The results reported here show that NSP3 forms a link between viral mRNA and the cellular translation machinery and hence is a functional analogue of cellular poly(A)-binding protein.
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Patel, Osman V., Anilkumar Bettegowda, James J. Ireland, Paul M. Coussens, Patrick Lonergan, and George W. Smith. "Functional genomics studies of oocyte competence: evidence that reduced transcript abundance for follistatin is associated with poor developmental competence of bovine oocytes." Reproduction 133, no. 1 (January 2007): 95–106. http://dx.doi.org/10.1530/rep.1.01123.
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Poor oocyte competence contributes to infertility in humans and livestock species. The molecular characteristics of such oocytes are generally unknown. Objectives of the present studies were to identify differences in RNA transcript abundance in oocytes and early embryos associated with reduced oocyte competence and development to the blastocyst stage. Microarray experiments were conducted using RNA isolated from germinal vesicle stage oocytes collected from adult versus prepubertal animals (model of poor oocyte competence). A total of 193 genes displaying greater mRNA abundance in adult oocytes and 223 genes displaying greater mRNA abundance in prepubertal oocytes were detected. Subsequent gene ontology analysis of microarray data revealed significant overrepresentation of transcripts encoding for genes in hormone secretion classification within adult oocytes and such genes were selected for further analysis. Real-time PCR experiments revealed greater abundance of mRNA for βA and βB subunits of inhibin/activin and follistatin, but not the α subunit in germinal vesicle stage oocytes collected from adult versus prepubertal animals. Cumulus cell follistatin and βB subunit mRNA abundance were similar in samples collected from prepubertal versus adult animals. A positive association between time of first cleavage (oocyte competence) and follistatin mRNA abundance was noted. Follistatin, βB, and α subunit mRNAs were temporally regulated during early bovine embryogenesis and peaked at the 16-cell stage. Collectively, results demonstrate a positive association of follistatin mRNA abundance with oocyte competence in two distinct models and dynamic regulation of follistatin, βB, and α subunit mRNAs in early embryos after initiation of transcription from the embryonic genome.
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Wellinger, Ralf E., Félix Prado, and Andrés Aguilera. "Replication Fork Progression Is Impaired by Transcription in Hyperrecombinant Yeast Cells Lacking a Functional THO Complex." Molecular and Cellular Biology 26, no. 8 (April 15, 2006): 3327–34. http://dx.doi.org/10.1128/mcb.26.8.3327-3334.2006.
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ABSTRACT THO/TREX is a conserved, eukaryotic protein complex operating at the interface between transcription and messenger ribonucleoprotein (mRNP) metabolism. THO mutations impair transcription and lead to increased transcription-associated recombination (TAR). These phenotypes are dependent on the nascent mRNA; however, the molecular mechanism by which impaired mRNP biogenesis triggers recombination in THO/TREX mutants is unknown. In this study, we provide evidence that deficient mRNP biogenesis causes slowdown or pausing of the replication fork in hpr1Δ mutants. Impaired replication appears to depend on sequence-specific features since it was observed upon activation of lacZ but not leu2 transcription. Replication fork progression could be partially restored by hammerhead ribozyme-guided self-cleavage of the nascent mRNA. Additionally, hpr1Δ increased the number of S-phase but not G2-dependent TAR events as well as the number of budded cells containing Rad52 repair foci. Our results link transcription-dependent genomic instability in THO mutants with impaired replication fork progression, suggesting a molecular basis for a connection between inefficient mRNP biogenesis and genetic instability.
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Zhang, Yandong, Jin You, Xingshun Wang, and Jason Weber. "The DHX33 RNA Helicase Promotes mRNA Translation Initiation." Molecular and Cellular Biology 35, no. 17 (June 22, 2015): 2918–31. http://dx.doi.org/10.1128/mcb.00315-15.
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DEAD/DEAH box RNA helicases play essential roles in numerous RNA metabolic processes, such as mRNA translation, pre-mRNA splicing, ribosome biogenesis, and double-stranded RNA sensing. Herein we show that a recently characterized DEAD/DEAH box RNA helicase, DHX33, promotes mRNA translation initiation. We isolated intact DHX33 protein/RNA complexes in cells and identified several ribosomal proteins, translation factors, and mRNAs. Reduction of DHX33 protein levels markedly reduced polyribosome formation and caused the global inhibition of mRNA translation that was rescued with wild-type DHX33 but not helicase-defective DHX33. Moreover, we observed an accumulation of mRNA complexes with the 80S ribosome in the absence of functional DHX33, consistent with a stalling in initiation, and DHX33 more preferentially promoted structured mRNA translation. We conclude that DHX33 functions to promote elongation-competent 80S ribosome assembly at the late stage of mRNA translation initiation. Our results reveal a newly recognized function of DHX33 in mRNA translation initiation, further solidifying its central role in promoting cell growth and proliferation.