Journal articles on the topic 'Nuclear speckles domain'
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1
Xie, Sheila Q., Sonya Martin, Pascale V. Guillot, David L. Bentley, and Ana Pombo. "Splicing Speckles Are Not Reservoirs of RNA Polymerase II, but Contain an Inactive Form, Phosphorylated on Serine2 Residues of the C-Terminal Domain." Molecular Biology of the Cell 17, no. 4 (April 2006): 1723–33. http://dx.doi.org/10.1091/mbc.e05-08-0726.
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“Splicing speckles” are major nuclear domains rich in components of the splicing machinery and polyA+ RNA. Although speckles contain little detectable transcriptional activity, they are found preferentially associated with specific mRNA-coding genes and gene-rich R bands, and they accumulate some unspliced pre-mRNAs. RNA polymerase II transcribes mRNAs and is required for splicing, with some reports suggesting that the inactive complexes are stored in splicing speckles. Using ultrathin cryosections to improve optical resolution and preserve nuclear structure, we find that all forms of polymerase II are present, but not enriched, within speckles. Inhibition of polymerase activity shows that speckles do not act as major storage sites for inactive polymerase II complexes but that they contain a stable pool of polymerase II phosphorylated on serine2 residues of the C-terminal domain, which is transcriptionally inactive and may have roles in spliceosome assembly or posttranscriptional splicing of pre-mRNAs. Paraspeckle domains lie adjacent to speckles, but little is known about their protein content or putative roles in the expression of the speckle-associated genes. We find that paraspeckles are transcriptionally inactive but contain polymerase II, which remains stably associated upon transcriptional inhibition, when paraspeckles reorganize around nucleoli in the form of caps.
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Schneider, Jana, Bianca Dauber, Krister Melén, Ilkka Julkunen, and Thorsten Wolff. "Analysis of Influenza B Virus NS1 Protein Trafficking Reveals a Novel Interaction with Nuclear Speckle Domains." Journal of Virology 83, no. 2 (November 5, 2008): 701–11. http://dx.doi.org/10.1128/jvi.01858-08.
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ABSTRACT Many proteins that function in the transcription, maturation, and export of metazoan mRNAs are concentrated in nuclear speckle domains, indicating that the compartment is important for gene expression. Here, we show that the NS1 protein of influenza B virus (B/NS1) accumulates in nuclear speckles and causes rounding and morphological changes of the domains, indicating a disturbance in their normal functions. This property was located within the N-terminal 90 amino acids of the B/NS1 protein and was shown to be independent of any other viral gene product. Within this protein domain, we identified a monopartite importin α binding nuclear localization signal. Reverse-genetic analysis of this motif indicated that nuclear import and speckle association of the B/NS1 protein are required for the full replication capacity of the virus. In the late phase of virus infection, the B/NS1 protein relocated to the cytoplasm, which occurred in a CRM1-independent manner. The interaction of the B/NS1 protein with nuclear speckles may reflect a recruitment function to promote viral-gene expression. To our knowledge, this is the first functional description of a speckle-associated protein that is encoded by a negative-strand RNA virus.
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Shakyawar, Dhruv Kumar, Bhattiprolu Muralikrishna, and Vegesna Radha. "C3G dynamically associates with nuclear speckles and regulates mRNA splicing." Molecular Biology of the Cell 29, no. 9 (May 2018): 1111–24. http://dx.doi.org/10.1091/mbc.e17-07-0442.
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C3G (Crk SH3 domain binding guanine nucleotide releasing factor) (Rap guanine nucleotide exchange factor 1), essential for mammalian embryonic development, is ubiquitously expressed and undergoes regulated nucleocytoplasmic exchange. Here we show that C3G localizes to SC35-positive nuclear speckles and regulates splicing activity. Reversible association of C3G with speckles was seen on inhibition of transcription and splicing. C3G shows partial colocalization with SC35 and is recruited to a chromatin and RNase-sensitive fraction of speckles. Its presence in speckles is dependent on intact cellular actin cytoskeleton and is lost on expression of the kinase Clk1. Rap1, a substrate of C3G, is also present in nuclear speckles, and inactivation of Rap signaling by expression of GFP-Rap1GAP alters speckle morphology and number. Enhanced association of C3G with speckles is seen on glycogen synthase kinase 3 beta inhibition or differentiation of C2C12 cells to myotubes. CRISPR/Cas9-mediated knockdown of C3G resulted in altered splicing activity of an artificial gene as well as endogenous CD44. C3G knockout clones of C2C12 as well as MDA-MB-231 cells showed reduced protein levels of several splicing factors compared with control cells. Our results identify C3G and Rap1 as novel components of nuclear speckles and a role for C3G in regulating cellular RNA splicing activity.
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Gama-Carvalho, Margarida, Randy D. Krauss, Lijian Chiang, Juan Valcárcel, Michael R. Green, and Maria Carmo-Fonseca. "Targeting of U2AF65 to Sites of Active Splicing in the Nucleus." Journal of Cell Biology 137, no. 5 (June 2, 1997): 975–87. http://dx.doi.org/10.1083/jcb.137.5.975.
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U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.
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Cáceres, Javier F., Tom Misteli, Gavin R. Screaton, David L. Spector, and Adrian R. Krainer. "Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity." Journal of Cell Biology 138, no. 2 (July 28, 1997): 225–38. http://dx.doi.org/10.1083/jcb.138.2.225.
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SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.
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Prasanth, Kannanganattu V., Matthew Camiolo, Grace Chan, Vidisha Tripathi, Laurence Denis, Tetsuya Nakamura, Michael R. Hübner, and David L. Spector. "Nuclear Organization and Dynamics of 7SK RNA in Regulating Gene Expression." Molecular Biology of the Cell 21, no. 23 (December 2010): 4184–96. http://dx.doi.org/10.1091/mbc.e10-02-0105.
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Noncoding RNAs play important roles in various aspects of gene regulation. We have identified 7SK RNA to be enriched in nuclear speckles or interchromatin granule clusters (IGCs), a subnuclear domain enriched in pre-mRNA processing factors. 7SK RNA, in association with HEXIM 1 and 2, is involved in the inhibition of transcriptional elongation by RNA polymerase II. Inhibition occurs via sequestration of the active P-TEFb kinase complex (CDK 9 and Cyclin T1/T2a/b or K) that is involved in phosphorylating the C-terminal domain of RNA polymerase II. Our results demonstrate that knock-down of 7SK RNA, by specific antisense oligonucleotides, results in the mislocalization of nuclear speckle constituents in a transcription-dependent manner, and the transcriptional up-regulation of a RNA polymerase II transcribed reporter gene locus. Furthermore, 7SK RNA transiently associates with a stably integrated reporter gene locus upon transcriptional down-regulation and its presence correlates with the efficient displacement of P-TEFb constituents from the locus. Our results suggest that 7SK RNA plays a role in modulating the available level of P-TEFb upon transcriptional down-regulation by sequestering its constituents in nuclear speckles.
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Burgute, Bhagyashri D., Vivek S. Peche, Anna-Lena Steckelberg, Gernot Glöckner, Berthold Gaßen, Niels H. Gehring, and Angelika A. Noegel. "NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins." Nucleic Acids Research 42, no. 5 (December 17, 2013): 3177–93. http://dx.doi.org/10.1093/nar/gkt1311.
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Abstract NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS–NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing.
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Misteli, Tom, Javier F. Cáceres, Jade Q. Clement, Adrian R. Krainer, Miles F. Wilkinson, and David L. Spector. "Serine Phosphorylation of SR Proteins Is Required for Their Recruitment to Sites of Transcription In Vivo." Journal of Cell Biology 143, no. 2 (October 19, 1998): 297–307. http://dx.doi.org/10.1083/jcb.143.2.297.
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Expression of most RNA polymerase II transcripts requires the coordinated execution of transcription, splicing, and 3′ processing. We have previously shown that upon transcriptional activation of a gene in vivo, pre-mRNA splicing factors are recruited from nuclear speckles, in which they are concentrated, to sites of transcription (Misteli, T., J.F. Cáceres, and D.L. Spector. 1997. Nature. 387:523–527). This recruitment process appears to spatially coordinate transcription and pre-mRNA splicing within the cell nucleus. Here we have investigated the molecular basis for recruitment by analyzing the recruitment properties of mutant splicing factors. We show that multiple protein domains are required for efficient recruitment of SR proteins from nuclear speckles to nascent RNA. The two types of modular domains found in the splicing factor SF2/ ASF exert distinct functions in this process. In living cells, the RS domain functions in the dissociation of the protein from speckles, and phosphorylation of serine residues in the RS domain is a prerequisite for this event. The RNA binding domains play a role in the association of splicing factors with the target RNA. These observations identify a novel in vivo role for the RS domain of SR proteins and suggest a model in which protein phosphorylation is instrumental for the recruitment of these proteins to active sites of transcription in vivo.
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Jagiello, I., A. Van Eynde, V. Vulsteke, M. Beullens, A. Boudrez, S. Keppens, W. Stalmans, and M. Bollen. "Nuclear and subnuclear targeting sequences of the protein phosphatase-1 regulator NIPP1." Journal of Cell Science 113, no. 21 (November 1, 2000): 3761–68. http://dx.doi.org/10.1242/jcs.113.21.3761.
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NIPP1 is a nuclear subunit of protein phosphatase-1 (PP1) that colocalizes with pre-mRNA splicing factors in speckles. We report here that the nuclear and subnuclear targeting of NIPP1, when expressed in HeLa cells or COS-1 cells as a fusion protein with the enhanced-green-fluorescent protein (EGFP), are mediated by distinct sequences. While NIPP1-EGFP can cross the nuclear membrane passively, the active transport to the nucleus is mediated by two independent nuclear localization signals in the central domain of NIPP1, which partially overlap with binding site(s) for PP1. Furthermore, the concentration of NIPP1-EGFP in the nuclear speckles requires the ‘ForkHead-Associated’ domain in the N terminus. This domain is also required for the nuclear retention of NIPP1 when active transport is blocked. Our data imply that the nuclear and subnuclear targeting of NIPP1 are controlled independently.
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Miyagi, Tamami, Rio Yamazaki, Koji Ueda, Satoshi Narumi, Yuhei Hayamizu, Hiroshi Uji-i, Masahiko Kuroda, and Kohsuke Kanekura. "The Patterning and Proportion of Charged Residues in the Arginine-Rich Mixed-Charge Domain Determine the Membrane-Less Organelle Targeted by the Protein." International Journal of Molecular Sciences 23, no. 14 (July 11, 2022): 7658. http://dx.doi.org/10.3390/ijms23147658.
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Membrane-less organelles (MLOs) are formed by biomolecular liquid–liquid phase separation (LLPS). Proteins with charged low-complexity domains (LCDs) are prone to phase separation and localize to MLOs, but the mechanism underlying the distributions of such proteins to specific MLOs remains poorly understood. Recently, proteins with Arg-enriched mixed-charge domains (R-MCDs), primarily composed of R and Asp (D), were found to accumulate in nuclear speckles via LLPS. However, the process by which R-MCDs selectively incorporate into nuclear speckles is unknown. Here, we demonstrate that the patterning of charged amino acids and net charge determines the targeting of specific MLOs, including nuclear speckles and the nucleolus, by proteins. The redistribution of R and D residues from an alternately sequenced pattern to uneven blocky sequences caused a shift in R-MCD distribution from nuclear speckles to the nucleolus. In addition, the incorporation of basic residues in the R-MCDs promoted their localization to the MLOs and their apparent accumulation in the nucleolus. The R-MCD peptide with alternating amino acids did not undergo LLPS, whereas the blocky R-MCD peptide underwent LLPS with affinity to RNA, acidic poly-Glu, and the acidic nucleolar protein nucleophosmin, suggesting that the clustering of R residues helps avoid their neutralization by D residues and eventually induces R-MCD migration to the nucleolus. Therefore, the distribution of proteins to nuclear speckles requires the proximal positioning of D and R for the mutual neutralization of their charges.
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Mortier, Eva, Gunther Wuytens, Iris Leenaerts, Femke Hannes, Man Y. Heung, Gisèle Degeest, Guido David, and Pascale Zimmermann. "Nuclear speckles and nucleoli targeting by PIP2–PDZ domain interactions." EMBO Journal 24, no. 14 (June 16, 2005): 2556–65. http://dx.doi.org/10.1038/sj.emboj.7600722.
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Ghosh, Arpita, Satya Prakash Pandey, Asgar Hussain Ansari, Jennifer Seematti Sundar, Praveen Singh, Yasmeen Khan, Mary Krishna Ekka, Debojyoti Chakraborty, and Souvik Maiti. "Alternative splicing modulation mediated by G-quadruplex structures in MALAT1 lncRNA." Nucleic Acids Research 50, no. 1 (November 11, 2021): 378–96. http://dx.doi.org/10.1093/nar/gkab1066.
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Abstract MALAT1, an abundant lncRNA specifically localized to nuclear speckles, regulates alternative-splicing (AS). The molecular basis of its role in AS remains poorly understood. Here, we report three conserved, thermodynamically stable, parallel RNA-G-quadruplexes (rG4s) present in the 3′ region of MALAT1 which regulates this function. Using rG4 domain-specific RNA-pull-down followed by mass-spectrometry, RNA-immuno-precipitation, and imaging, we demonstrate the rG4 dependent localization of Nucleolin (NCL) and Nucleophosmin (NPM) to nuclear speckles. Specific G-to-A mutations that abolish rG4 structures, result in the localization loss of both the proteins from speckles. Functionally, disruption of rG4 in MALAT1 phenocopies NCL knockdown resulting in altered pre-mRNA splicing of endogenous genes. These results reveal a central role of rG4s within the 3′ region of MALAT1 orchestrating AS.
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Zhou, Anyu, Aeri Cho, Shu-Ching Huang, and Edward J. Benz. "A Novel Splicing Factor RBM25 in Erythroid Differentiation." Blood 110, no. 11 (November 16, 2007): 1711. http://dx.doi.org/10.1182/blood.v110.11.1711.1711.
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Abstract Many protein factors that guide pre-mRNA modification pathways are composed of RNA recognition motif (RRM) domains. In an effort to identify splicing regulators in erythroid cells, we cloned a RRM-containing protein RBM25 and characterized its role in alternative splicing. RBM25 consists of a proline-rich region and a RRM domain at the amino-terminal end, an ER rich domain at the central region, and a PWI domain at the carboxyl terminal end. RBM25 partially co-localized with splicing factor SC35 in nuclear speckles. While both the RRM and PWI domains are diffusely distributed in the nucleoplasm, the ER domain is highly concentrated in nuclear speckles. We examined cellular mRNA targets of RBM25 in HeLa cells and demonstrated that it binds Bcl-x mRNA and affects its alternative splicing. Depletion of RBM25 by RNA interference caused accumulation of anti-apoptotic Bcl-x(L), whereas its up-regulation increased the levels of pro-apoptotic Bcl-x(s). The expression level of RBM25 also correlated with the degree of cell death, further suggesting that it functions in regulating apoptotic factor(s) expression. Apoptosis plays an important role in red cell development; earlier erythroid progenitors are more sensitive to apoptosis while mature erythroblasts are resistant to apoptosis. A significant decrease in RBM25 expression occurs during erythroid differentiation and correlates with resistance to apoptosis. Our results suggest that erythroblasts may acquire resistance to apoptosis during maturation through differential expression of crucial splicing regulators of the apoptotic machinery.
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Herrmann, C. H., and M. A. Mancini. "The Cdk9 and cyclin T subunits of TAK/P-TEFb localize to splicing factor-rich nuclear speckle regions." Journal of Cell Science 114, no. 8 (April 15, 2001): 1491–503. http://dx.doi.org/10.1242/jcs.114.8.1491.
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TAK/P-TEFb is an elongation factor for RNA polymerase II-directed transcription that is thought to function by phosphorylating the C-terminal domain of the largest subunit of RNA polymerase II. TAK/P-TEFb is composed of Cdk9 and cyclin T and serves as the cellular cofactor for the human immunodeficiency virus transactivator Tat protein. In this study, we examined the subcellular distribution of Cdk9 and cyclin T1 using high resolution immunofluorescence microscopy and found that Cdk9 and cyclin T1 localized throughout the non-nucleolar nucleoplasm, with increased signal present at numerous foci. Both Cdk9 and cyclin T1 showed only limited colocalization with different phosphorylated forms of RNA polymerase II. However, significant colocalization with antibodies to several splicing factors that identify nuclear ‘speckles’ was observed for Cdk9 and especially for cyclin T1. The pattern of Cdk9 and cyclin T1 distribution was altered in cells treated with transcription inhibitors. Transient expression of cyclin T1 deletion mutants indicated that a region in the central portion of cyclin T1 is important for accumulation at speckles. Furthermore, cyclin T1 proteins that accumulated at speckles were capable of recruiting Cdk9 and the HIV Tat protein to this compartment in overexpression experiments. These results suggest that cyclin T1 functions to recruit its binding partners to nuclear speckles and raises the possibility that nuclear speckles are a site of TAK/P-TEFb function.
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Taylor, S. E., J. Bagnall, D. Mason, R. Levy, D. G. Fernig, and V. See. "Differential sub-nuclear distribution of hypoxia-inducible factors (HIF)-1 and -2 alpha impacts on their stability and mobility." Open Biology 6, no. 9 (September 2016): 160195. http://dx.doi.org/10.1098/rsob.160195.
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Cellular adaptation to hypoxia occurs via a complex programme of gene expression mediated by the hypoxia-inducible factor (HIF). The oxygen labile alpha subunits, HIF-1α/-2α, form a heterodimeric transcription factor with HIF-1β and modulate gene expression. HIF-1α and HIF-2α possess similar domain structure and bind to the same consensus sequence. However, they have different oxygen-dependent stability and activate distinct genes. To better understand these differences, we used fluorescent microscopy to determine precise localization and dynamics. We observed a homogeneous distribution of HIF-1α in the nucleus, while HIF-2α localized into speckles. We demonstrated that the number, size and mobility of HIF-2α speckles were independent of cellular oxygenation and that HIF-2α molecules were capable of exchanging between the speckles and nucleoplasm in an oxygen-independent manner. The concentration of HIF-2α into speckles may explain its increased stability compared with HIF-1α and its slower mobility may offer a mechanism for gene specificity.
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Schwam, David R., Randy L. Luciano, Shahana S. Mahajan, LaiYee Wong, and Angus C. Wilson. "Carboxy Terminus of Human Herpesvirus 8 Latency-Associated Nuclear Antigen Mediates Dimerization, Transcriptional Repression, and Targeting to Nuclear Bodies." Journal of Virology 74, no. 18 (September 15, 2000): 8532–40. http://dx.doi.org/10.1128/jvi.74.18.8532-8540.2000.
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ABSTRACT Human herpesvirus 8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus) is the causative agent of Kaposi's sarcoma and certain B-cell lymphomas. In most infected cells, HHV-8 establishes a latent infection characterized by the expression of latency-associated nuclear antigen (LANA) encoded by open reading frame 73. Although unrelated by sequence, there are functional similarities between LANA and the EBNA-1 protein of Epstein-Barr virus. Both accumulate as subnuclear speckles and are required for maintenance of the viral episome. EBNA-1 also regulates viral gene expression and is required for cell immortalization, suggesting that LANA performs similar functions in the context of HHV-8 infection. Here we show that LANA forms stable dimers, or possibly higher-order multimers, and that this is mediated by a conserved region in the C terminus. By expressing a series of truncations, we show that both the N- and C-terminal regions localize to the nucleus, although only the C terminus accumulates as nuclear speckles characteristic of the intact protein. Lastly, we show that LANA can function as a potent transcriptional repressor when tethered to constitutively active promoters via a heterologous DNA-binding domain. Domains in both the N and C termini mediate repression. This suggests that one function of LANA is to suppress the expression of the viral lytic genes or cellular genes involved in the antiviral response.
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LLORIAN, Miriam, Monique BEULLENS, Isabel ANDRÉS, Jose-Miguel ORTIZ, and Mathieu BOLLEN. "SIPP1, a novel pre-mRNA splicing factor and interactor of protein phosphatase-1." Biochemical Journal 378, no. 1 (February 15, 2004): 229–38. http://dx.doi.org/10.1042/bj20030950.
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We have identified a polypeptide that was already known to interact with polyglutamine-tract-binding protein (PQBP)-1/Npw38 as a novel splicing factor and interactor of protein phosphatase-1, hence the name SIPP1 for splicing factor that interacts with PQBP-1 and PP1 (protein phosphotase 1). SIPP1 was inhibitory to PP1, and its inhibitory potency was increased by phosphorylation with protein kinase CK1. Two-hybrid and co-sedimentation analysis revealed that SIPP1 has two distinct PP1-binding domains and that the binding of SIPP1 with PP1 involves a RVXF (Arg-Val-Xaa-Phe) motif, which functions as a PP1-binding sequence in most interactors of PP1. Enhanced-green-fluorescent-protein-tagged SIPP1 was targeted exclusively to the nucleus and was enriched in the nuclear speckles, which represent storage/assembly sites of splicing factors. We have mapped a nuclear localization signal in the N-terminus of SIPP1, while the proline-rich C-terminal domain appeared to be required for its subnuclear targeting to the speckles. Finally, we found that SIPP1 is also a component of the spliceosomes and that a SIPP1-fragment inhibits splicing catalysis by nuclear extracts independent of its ability to interact with PP1.
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Simone, Federico, Paul E. Polak, Joseph J. Kaberlein, Roger T. Luo, Denise A. Levitan, and Michael J. Thirman. "EAF1, a novel ELL-associated factor that is delocalized by expression of the MLL-ELL fusion protein." Blood 98, no. 1 (July 1, 2001): 201–9. http://dx.doi.org/10.1182/blood.v98.1.201.
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Abstract The (11;19)(q23;p13.1) translocation in acute leukemia leads to the generation of a chimeric protein that fuses MLL to the transcriptional elongation factor ELL. A novel protein was isolated from a yeast 2-hybrid screen with ELL that was named EAF1 for ELL-associated factor 1. Using specific antibodies, the endogenous EAF1 and ELL proteins were coimmunoprecipitated from multiple cell lines. In addition, endogenous EAF1 also exhibited the capacity to interact with ELL2. Database comparisons with EAF1 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that exhibited homology with the transcriptional activation domains of several translocation partner proteins of MLL, including AF4, LAF4, and AF5q31. A similar transcriptional activation domain has been identified in this region of EAF1. By confocal microscopy, endogenous EAF1 and ELL colocalized in a distinct nuclear speckled pattern. Transfection of theMLL-ELL fusion gene delocalized EAF1 from its nuclear speckled distribution to a diffuse nucleoplasmic pattern. In leukemic cell lines derived from mice transplanted withMLL-ELL–transduced bone marrow, EAF1 speckles were not detected. Taken together, these data suggest that expression of the MLL-ELL fusion protein may have a dominant effect on the normal protein-protein interactions of ELL.
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SEMPLE, Jennifer I., Stephanie E. BROWN, Christopher M. SANDERSON, and R. Duncan CAMPBELL. "A distinct bipartite motif is required for the localization of inhibitory κB-like (IκBL) protein to nuclear speckles." Biochemical Journal 361, no. 3 (January 25, 2002): 489–96. http://dx.doi.org/10.1042/bj3610489.
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The inhibitory κB (IκB)-like (IκBL) gene is located within the Class III region of the MHC on human chromosome 6. Previous analysis of the predicted amino acid sequence of the human IκBL protein revealed three putative functional domains; 2–3 ankyrin repeat sequences, which are similar to the second and third ankyrin repeats of the nuclear factor κB (NF-κB) protein; three PEST sequence motifs (a sequence that is rich in proline, serine, aspartic acid and threonine residues), which are also found in other IκB family members; and a C-terminal leucine zipper-like motif. In the present study we have identified a novel bipartite motif, which is required for nuclear localization of the IκBL protein. Analyses of IκBL-specific transcripts revealed the existence of a widely expressed spliced variant form of IκBL (IκBLsv1), which lacks the amino acid sequence GELEDEWQEVMGRFE (where single-letter amino-acid notation has been used). Interestingly, translation of IκBL mRNA in vivo was found to initiate predominantly from the second available methionine, thereby resulting in the disruption of the predicted N-terminal PEST sequence. Also, transient expression of T7 epitope-tagged IκBL and IκBLsv1 proteins in mammalian cells showed that both proteins were targeted to the nucleus, where they accumulate in nuclear speckles. To define the protein domains required for nuclear import and subnuclear localization, a complementary set of deletion mutants and enhanced green fluorescent protein—IκBL domain fusions were expressed in mammalian cells. Data from these experiments show that a combination of the ankyrin-repeat region and an adjacent arginine-rich sequence are necessary and sufficient for both nuclear import and speckle localization.
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Elong Edimo, William's, Rita Derua, Veerle Janssens, Takeshi Nakamura, Jean-Marie Vanderwinden, Etienne Waelkens, and Christophe Erneux. "Evidence of SHIP2 Ser132 phosphorylation, its nuclear localization and stability." Biochemical Journal 439, no. 3 (October 13, 2011): 391–404. http://dx.doi.org/10.1042/bj20110173.
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PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are major signalling molecules in mammalian cell biology. PtdIns(3,4)P2 can be produced by PI3Ks [PI (phosphoinositide) 3-kinases], but also by PI 5-phosphatases including SHIP2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2]. Proteomic studies in human cells revealed that SHIP2 can be phosphorylated at more than 20 sites, but their individual function is unknown. In a model of PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null astrocytoma cells, lowering SHIP2 expression leads to increased PtdIns(3,4,5)P3 levels and Akt phosphorylation. MS analysis identified SHIP2 phosphosites on Ser132, Thr1254 and Ser1258; phosphotyrosine-containing sites were undetectable. By immunostaining, total SHIP2 concentrated in the perinuclear area and in the nucleus, whereas SHIP2 phosphorylated on Ser132 was in the cytoplasm, the nucleus and nuclear speckles, depending on the cell cycle stage. SHIP2 phosphorylated on Ser132 demonstrated PtdIns(4,5)P2 phosphatase activity. Endogenous phospho-SHIP2 (Ser132) showed an overlap with PtdIns(4,5)P2 staining in nuclear speckles. SHIP2 S132A was less sensitive to C-terminal degradation and more resistant to calpain as compared with wild-type enzyme. We have identified nuclear lamin A/C as a novel SHIP2 interactor. We suggest that the function of SHIP2 is different at the plasma membrane where it recognizes PtdIns(3,4,5)P3, and in the nucleus where it may interact with PtdIns(4,5)P2, particularly in speckles.
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Terada, Yasuhiko, and Yuko Yasuda. "Human Immunodeficiency Virus Type 1 Vpr Induces G2 Checkpoint Activation by Interacting with the Splicing Factor SAP145." Molecular and Cellular Biology 26, no. 21 (August 21, 2006): 8149–58. http://dx.doi.org/10.1128/mcb.01170-06.
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ABSTRACT Vpr, the viral protein R of human immunodeficiency virus type 1, induces G2 cell cycle arrest and apoptosis in mammalian cells via ATR (for “ataxia-telangiectasia-mediated and Rad3-related”) checkpoint activation. The expression of Vpr induces the formation of the γ-histone 2A variant X (H2AX) and breast cancer susceptibility protein 1 (BRCA1) nuclear foci, and a C-terminal domain is required for Vpr-induced ATR activation and its nuclear localization. However, the cellular target of Vpr, as well as the mechanism of G2 checkpoint activation, was unknown. Here we report that Vpr induces checkpoint activation and G2 arrest by binding to the CUS1 domain of SAP145 and interfering with the functions of the SAP145 and SAP49 proteins, two subunits of the multimeric splicing factor 3b (SF3b). Vpr interacts with and colocalizes with SAP145 through its C-terminal domain in a speckled distribution. The depletion of either SAP145 or SAP49 leads to checkpoint-mediated G2 cell cycle arrest through the induction of nuclear foci containing γ-H2AX and BRCA1. In addition, the expression of Vpr excludes SAP49 from the nuclear speckles and inhibits the formation of the SAP145-SAP49 complex. To conclude, these results point out the unexpected roles of the SAP145-SAP49 splicing factors in cell cycle progression and suggest that cellular expression of Vpr induces checkpoint activation and G2 arrest by interfering with the function of SAP145-SAP49 complex in host cells.
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Bridger, J. M., H. Herrmann, C. Munkel, and P. Lichter. "Identification of an interchromosomal compartment by polymerization of nuclear-targeted vimentin." Journal of Cell Science 111, no. 9 (May 1, 1998): 1241–53. http://dx.doi.org/10.1242/jcs.111.9.1241.
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A number of structural and functional subnuclear compartments have been described, including regions exclusive of chromosomes previously hypothesized to form a reactive nuclear space. We have now explored this accessible nuclear space and interchromosomal nucleoplasmic domains experimentally using Xenopus vimentin engineered to contain a nuclear localization signal (NLS-vimentin). In stably transfected human cells incubated at 37 degrees C, the NLS-vimentin formed a restricted number of intranuclear speckles. At 28 degrees C, the optimal temperature for assembly of the amphibian protein, NLS-vimentin progressively extended with time out from the speckles into strictly orientated intranuclear filamentous arrays. This enabled us to observe the development of a system of interconnecting channel-like areas. Quantitative analysis based on 3-D imaging microscopy revealed that these arrays were localized almost exclusively outside of chromosome territories. During mitosis the filaments disassembled and dispersed throughout the cytoplasm, while in anaphase-telophase the vimentin was recruited back into the nucleus and reassembled into filaments at the chromosome surfaces, in distributions virtually identical to those observed in the previous interphase. The filaments also colocalized with specific nuclear RNAs, coiled bodies and PML bodies, all situated outside of chromosome territories, thereby interlinking these structures. This strongly implies that these nuclear entities coexist in the same interconnected nuclear compartment. The assembling NLS-vimentin is restricted to and can be used to delineate, at least in part, the formerly proposed reticular interchromosomal domain compartment (ICD). The properties of NLS-vimentin make it an excellent tool for performing structural and functional studies on this compartment.
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Sánchez-Álvarez, Miguel, Aaron C. Goldstrohm, Mariano A. Garcia-Blanco, and Carlos Suñé. "Human Transcription Elongation Factor CA150 Localizes to Splicing Factor-Rich Nuclear Speckles and Assembles Transcription and Splicing Components into Complexes through Its Amino and Carboxyl Regions." Molecular and Cellular Biology 26, no. 13 (July 1, 2006): 4998–5014. http://dx.doi.org/10.1128/mcb.01991-05.
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ABSTRACT The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while WW domains are not required for colocalization. We also performed biochemical assays to understand the role of WW and FF domains in mediating the assembly of transcription and splicing components into higher-order complexes. Transcription and splicing components bound to a region in the amino-terminal part of CA150 that contains the three WW domains; however, we identified a region of the C-terminal FF domains that was also critical. Our results suggest that sequences located at both the amino and carboxyl regions of CA150 are required to assemble transcription/splicing complexes, which may be involved in the coupling of those processes.
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Divecha, Nullin. "Phosphoinositides in the nucleus and myogenic differentiation: how a nuclear turtle with a PHD builds muscle." Biochemical Society Transactions 44, no. 1 (February 9, 2016): 299–306. http://dx.doi.org/10.1042/bst20150238.
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Phosphoinositides are a family of phospholipid messenger molecules that control various aspects of cell biology in part by interacting with and regulating downstream protein partners. Importantly, phosphoinositides are present in the nucleus. They form part of the nuclear envelope and are present within the nucleus in nuclear speckles, intra nuclear chromatin domains, the nuclear matrix and in chromatin. What their exact role is within these compartments is not completely clear, but the identification of nuclear specific proteins that contain phosphoinositide interaction domains suggest that they are important regulators of DNA topology, chromatin conformation and RNA maturation and export. The plant homeo domain (PHD) finger is a phosphoinositide binding motif that is largely present in nuclear proteins that regulate chromatin conformation. In the present study I outline how changes in the levels of the nuclear phosphoinositide PtdIns5P impact on muscle cell differentiation through the PHD finger of TAF3 (TAF, TATA box binding protein (TBP)-associated factor), which is a core component of a number of different basal transcription complexes.
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Patturajan, Meera, Xiangyun Wei, Ronald Berezney, and Jeffry L. Corden. "A Nuclear Matrix Protein Interacts with the Phosphorylated C-Terminal Domain of RNA Polymerase II." Molecular and Cellular Biology 18, no. 4 (April 1, 1998): 2406–15. http://dx.doi.org/10.1128/mcb.18.4.2406.
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ABSTRACT Yeast two-hybrid screening has led to the identification of a family of proteins that interact with the repetitive C-terminal repeat domain (CTD) of RNA polymerase II (A. Yuryev et al., Proc. Natl. Acad. Sci. USA 93:6975–6980, 1996). In addition to serine/arginine-rich SR motifs, the SCAFs (SR-like CTD-associated factors) contain discrete CTD-interacting domains. In this paper, we show that the CTD-interacting domain of SCAF8 specifically binds CTD molecules phosphorylated on serines 2 and 5 of the consensus sequence Tyr1Ser2Pro3Thr4Ser5Pro6Ser7. In addition, we demonstrate that SCAF8 associates with hyperphosphorylated but not with hypophosphorylated RNA polymerase II in vitro and in vivo. This result suggests that SCAF8 is not present in preinitiation complexes but rather associates with elongating RNA polymerase II. Immunolocalization studies show that SCAF8 is present in granular nuclear foci which correspond to sites of active transcription. We also provide evidence that SCAF8 foci are associated with the nuclear matrix. A fraction of these sites overlap with a subset of larger nuclear speckles containing phosphorylated polymerase II. Taken together, our results indicate a possible role for SCAF8 in linking transcription and pre-mRNA processing.
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Kumar, Priyadarsina, Louise Lindberg, Twanda L. Thirkill, Jennifer W. Ji, Lindsay Martsching, and Gordon C. Douglas. "The MUC1 Extracellular Domain Subunit Is Found in Nuclear Speckles and Associates with Spliceosomes." PLoS ONE 7, no. 8 (August 8, 2012): e42712. http://dx.doi.org/10.1371/journal.pone.0042712.
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Schmidt-Zachmann, Marion S., Sylvia Knecht, and Angela Krämer. "Molecular Characterization of a Novel, Widespread Nuclear Protein That Colocalizes with Spliceosome Components." Molecular Biology of the Cell 9, no. 1 (January 1998): 143–60. http://dx.doi.org/10.1091/mbc.9.1.143.
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We report the identification and molecular characterization of a novel type of constitutive nuclear protein that is present in diverse vertebrate species, from Xenopus laevis to human. The cDNA-deduced amino acid sequence of the Xenopus protein defines a polypeptide of a calculated mass of 146.2 kDa and a isoelectric point of 6.8, with a conspicuous domain enriched in the dipeptide TP (threonine-proline) near its amino terminus. Immunolocalization studies in cultured cells and tissues sections of different origin revealed an exclusive nuclear localization of the protein. The protein is diffusely distributed in the nucleoplasm but concentrated in nuclear speckles, which represent a subnuclear compartment enriched in small nuclear ribonucleoprotein particles and other splicing factors, as confirmed by colocalization with certain splicing factors and Sm proteins. During mitosis, when transcription and splicing are downregulated, the protein is released from the nuclear speckles and transiently dispersed throughout the cytoplasm. Biochemical experiments have shown that the protein is recovered in a ∼12S complex, and gel filtration studies confirm that the protein is part of a large particle. Immunoprecipitation and Western blot analysis of chromatographic fractions enriched in human U2 small nuclear ribonucleoprotein particles of distinct sizes (12S, 15S, and 17S), reflecting their variable association with splicing factors SF3a and SF3b, strongly suggests that the 146-kDa protein reported here is a constituent of the SF3b complex.
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Lawrence, Jeanne B., and Christine M. Clemson. "Gene associations: true romance or chance meeting in a nuclear neighborhood?" Journal of Cell Biology 182, no. 6 (September 22, 2008): 1035–38. http://dx.doi.org/10.1083/jcb.200808121.
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Many recent studies have raised interest in the nuclear associations of coregulated genes from different chromosomes, often evoking interpretations of gene–gene interactions, communication, and even “romance.” However, in some cases, the associations may be indirect and infrequent and may reflect the segregation of active and inactive genes into different nuclear compartments. The study by Brown et al. (see p. 1083 of this issue) reports that the apparent association of erythroid genes is not a direct interaction nor colocalization to one tiny transcription factory but arises as a result of the known clustering of many active genes with larger splicing factor–rich speckles (a.k.a., SC35-defined domains). This clustering appears largely stochastic but is impacted by the chromosomal neighborhood of the gene as well as its transcriptional status. The study adds a new twist by examining the same gene in a foreign chromosomal context, providing evidence that this impacts a gene's propensity to form gene–domain (or apparent gene–gene) associations within nuclei.
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Pendergrast, P. Shannon, Chen Wang, Nouria Hernandez, and Sui Huang. "FBI-1 Can Stimulate HIV-1 Tat Activity and Is Targeted to a Novel Subnuclear Domain that Includes the Tat-P-TEFb—containing Nuclear Speckles." Molecular Biology of the Cell 13, no. 3 (March 2002): 915–29. http://dx.doi.org/10.1091/mbc.01-08-0383.
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FBI-1 is a cellular POZ-domain–containing protein that binds to the HIV-1 LTR and associates with the HIV-1 transactivator protein Tat. Here we show that elevated levels of FBI-1 specifically stimulate Tat activity and that this effect is dependent on the same domain of FBI-1 that mediates Tat-FBI-1 association in vivo. FBI-1 also partially colocalizes with Tat and Tat's cellular cofactor, P-TEFb (Cdk9 and cyclin T1), at the splicing-factor–rich nuclear speckle domain. Further, a less-soluble population of FBI-1 distributes in a novel peripheral-speckle pattern of localization as well as in other nuclear regions. This distribution pattern is dependent on the FBI-1 DNA binding domain, on the presence of cellular DNA, and on active transcription. Taken together, these results suggest that FBI-1 is a cellular factor that preferentially associates with active chromatin and that can specifically stimulate Tat-activated HIV-1 transcription.
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Fraser, Kathryn A., and Stephen A. Rice. "Herpes Simplex Virus Type 1 Infection Leads to Loss of Serine-2 Phosphorylation on the Carboxyl-Terminal Domain of RNA Polymerase II." Journal of Virology 79, no. 17 (September 1, 2005): 11323–34. http://dx.doi.org/10.1128/jvi.79.17.11323-11334.2005.
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ABSTRACT Previous studies have shown that herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAP II), creating a new form of the enzyme known as RNAP III. However, the specific phosphorylation changes induced by HSV-1 have not been characterized. In this study, we used phospho-specific anti-CTD antibodies to probe the structure of the postinfection RNAP II. We find that RNAP III is phosphorylated on serine-5 (Ser-5) of the CTD consensus repeat but generally lacks phosphorylation on serine-2 (Ser-2). Since Ser-2 phosphorylation is normally associated with efficient transcriptional elongation and the recruitment of pre-mRNA processing factors, our results suggest that RNAP III may have altered elongation properties and decreased interactions with the mRNA processing machinery. The viral factors responsible for the reduction in Ser-2 CTD phosphorylation were studied. We found that viral immediate-early (IE) gene expression is required and sufficient, in the context of infection, for loss of Ser-2 phosphorylation. However, studies with viral mutants failed to implicate a single IE protein (among ICP0, ICP4, ICP22, and ICP27) in this process. Although most Ser-2-phosphorylated RNAP II is lost after infection, our immunofluorescence analyses identified a small subfraction that escapes loss and relocalizes to splicing antigen-rich nuclear speckles. A similar phenomenon is seen in uninfected cells after various treatments that inhibit RNAP II transcription. We hypothesize that the HSV-1-induced relocalization of residual Ser-2-phosphorylated RNAP II to nuclear speckles reflects a host response to the inhibition of cellular gene transcription.
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Nayler, Oliver, Annette M. Hartmann, and Stefan Stamm. "The ER Repeat Protein Yt521-B Localizes to a Novel Subnuclear Compartment." Journal of Cell Biology 150, no. 5 (September 4, 2000): 949–62. http://dx.doi.org/10.1083/jcb.150.5.949.
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The characterization of distinct subnuclear domains suggests a dynamic nuclear framework supporting gene expression and DNA replication. Here, we show that the glutamic acid/arginine-rich domain protein YT521-B localizes to a novel subnuclear structure, the YT bodies. YT bodies are dynamic compartments, which first appear at the beginning of S-phase in the cell cycle and disperse during mitosis. Furthermore, in untreated cells of the human cell line MCF7 they were undetectable and appeared only after drug- induced differentiation. YT bodies contain transcriptionally active sites and are in close contact to other subnuclear structures such as speckles and coiled bodies. YT bodies disperse upon actinomycin D treatment, whereas other transcriptional inhibitors such as α-amanitin or DRB have little effect. On the basis of our experiments, we propose that YT521-B may participate in the assembly of genes into transcription centers, thereby allowing efficient regulation of gene expression.
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Allemand, Eric, Svetlana Dokudovskaya, Rémy Bordonné, and Jamal Tazi. "A Conserved DrosophilaTransportin-Serine/Arginine-rich (SR) Protein Permits Nuclear Import ofDrosophila SR Protein Splicing Factors and Their Antagonist Repressor Splicing Factor 1." Molecular Biology of the Cell 13, no. 7 (July 2002): 2436–47. http://dx.doi.org/10.1091/mbc.e02-02-0102.
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Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted us to examine the mechanism by whichDrosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. Herein, we report the identification and characterization of a Drosophilaimportin β-family protein (dTRN-SR), homologous to TRN-SR2, that specifically interacts with both SR proteins and RSF1. dTRN-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with dTRN-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, we demonstrate that complementation with dTRN-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans.
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Barutcu, A. Rasim, Mingkun Wu, Ulrich Braunschweig, Boris J. A. Dyakov, Zheng Luo, Kyle M. Turner, Tanja Durbic, et al. "Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns." Molecular Cell 82, no. 5 (March 2022): 1035–52. http://dx.doi.org/10.1016/j.molcel.2021.12.010.
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LAI, Ming-Chih, Ru-Inn LIN, and Woan-Yuh TARN. "Differential effects of hyperphosphorylation on splicing factor SRp55." Biochemical Journal 371, no. 3 (May 1, 2003): 937–45. http://dx.doi.org/10.1042/bj20021827.
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Members of the serine/arginine-rich (SR) protein family play an important role in both constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich domain (RS domain) can modulate the activity and the subcellular localization of SR proteins. However, whether the SR protein family members are individually regulated and how this is achieved remain unclear. In this report we show that 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II-dependent transcription, specifically induced hyperphosphorylation of SRp55 but not that of any other SR proteins tested. Hyperphosphorylation of SRp55 occurs at the RS domain and appears to require the RNA-binding activity. Upon DRB treatment, hyperphosphorylated SRp55 relocates to enlarged nuclear speckles. Intriguingly, SRp55 is specifically targeted for degradation by the proteasome upon overexpression of the SR protein kinase Clk/Sty. Although a destabilization signal is mapped within the C-terminal 43-amino acid segment of SRp55, its adjacent lysine/serine-rich RS domain is nevertheless critical for the Clk/Sty-mediated degradation. We report for the first time that SRp55 can be hyperphosphorylated under different circumstances whereby its fate is differentially influenced.
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Hellert, Jan, Magdalena Weidner-Glunde, Joern Krausze, Heinrich Lünsdorf, Christiane Ritter, Thomas F. Schulz, and Thorsten Lührs. "The 3D structure of Kaposi sarcoma herpesvirus LANA C-terminal domain bound to DNA." Proceedings of the National Academy of Sciences 112, no. 21 (May 6, 2015): 6694–99. http://dx.doi.org/10.1073/pnas.1421804112.
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Kaposi sarcoma herpesvirus (KSHV) persists as a latent nuclear episome in dividing host cells. This episome is tethered to host chromatin to ensure proper segregation during mitosis. For duplication of the latent genome, the cellular replication machinery is recruited. Both of these functions rely on the constitutively expressed latency-associated nuclear antigen (LANA) of the virus. Here, we report the crystal structure of the KSHV LANA DNA-binding domain (DBD) in complex with its high-affinity viral target DNA, LANA binding site 1 (LBS1), at 2.9 Å resolution. In contrast to homologous proteins such as Epstein-Barr virus nuclear antigen 1 (EBNA-1) of the related γ-herpesvirus Epstein-Barr virus, specific DNA recognition by LANA is highly asymmetric. In addition to solving the crystal structure, we found that apart from the two known LANA binding sites, LBS1 and LBS2, LANA also binds to a novel site, denoted LBS3. All three sites are located in a region of the KSHV terminal repeat subunit previously recognized as a minimal replicator. Moreover, we show that the LANA DBD can coat DNA of arbitrary sequence by virtue of a characteristic lysine patch, which is absent in EBNA-1 of the Epstein-Barr virus. Likely, these higher-order assemblies involve the self-association of LANA into supermolecular spirals. One such spiral assembly was solved as a crystal structure of 3.7 Å resolution in the absence of DNA. On the basis of our data, we propose a model for the controlled nucleation of higher-order LANA oligomers that might contribute to the characteristic subnuclear KSHV microdomains (“LANA speckles”), a hallmark of KSHV latency.
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Chkheidze, Alexander N., and Stephen A. Liebhaber. "A Novel Set of Nuclear Localization Signals Determine Distributions of the αCP RNA-Binding Proteins." Molecular and Cellular Biology 23, no. 23 (December 1, 2003): 8405–15. http://dx.doi.org/10.1128/mcb.23.23.8405-8415.2003.
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ABSTRACT αCPs comprise a subfamily of KH-domain-containing RNA-binding proteins with specificity for C-rich pyrimidine tracts. These proteins play pivotal roles in a broad spectrum of posttranscriptional events. The five major αCP isoforms are encoded by four dispersed loci. Each isoform contains three repeats of the RNA-binding KH domain (KH1, KH2, and KH3) but lacks other identifiable motifs. To explore the complexity of their respective functions, we examined the subcellular localization of each αCP isoform. Immunofluorescence studies revealed three distinct distributions: αCP1 and αCP2 are predominantly nuclear with specific enrichment of αCP1 in nuclear speckles, αCP3 and αCP4 are restricted to the cytoplasm, and αCP2-KL, an αCP2 splice variant, is present at significant levels in both the nucleus and the cytoplasm. We mapped nuclear localization signals (NLSs) for αCP isoforms. αCP2 contains two functionally independent NLS. Both NLSs appear to be novel and were mapped to a 9-amino-acid segment between KH2 and KH3 (NLS I) and to a 12-amino-acid segment within KH3 (NLS II). NLS I is conserved in αCP1, whereas NLS II is inactivated by two amino acid substitutions. Neither NLS is present in αCP3 or αCP4. Consistent with mapping studies, deletion of NLS I from αCP1 blocks its nuclear accumulation, whereas NLS I and NLS II must both be inactivated to block nuclear accumulation of αCP2. These data demonstrate an unexpected complexity in the compartmentalization of αCP isoforms and identify two novel NLS that play roles in their respective distributions. This complexity of αCP distribution is likely to contribute to the diverse functions mediated by this group of abundant RNA-binding proteins.
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Tripathi, Vidisha, David Y. Song, Xinying Zong, Sergey P. Shevtsov, Stephen Hearn, Xiang-Dong Fu, Miroslav Dundr, and Kannanganattu V. Prasanth. "SRSF1 regulates the assembly of pre-mRNA processing factors in nuclear speckles." Molecular Biology of the Cell 23, no. 18 (September 15, 2012): 3694–706. http://dx.doi.org/10.1091/mbc.e12-03-0206.
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The mammalian cell nucleus is compartmentalized into nonmembranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and noncoding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long noncoding metastasis-associated lung adenocarcinoma transcript 1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role of SRSF1 in RNA polymerase II–mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus.
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Sakashita, Eiji, Sawako Tatsumi, Dieter Werner, Hitoshi Endo, and Akila Mayeda. "Human RNPS1 and Its Associated Factors: a Versatile Alternative Pre-mRNA Splicing Regulator In Vivo." Molecular and Cellular Biology 24, no. 3 (February 1, 2004): 1174–87. http://dx.doi.org/10.1128/mcb.24.3.1174-1187.2004.
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ABSTRACT Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently. To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library. Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2β (hTra2β; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles). The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2β, respectively. Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo. Overexpression of RNPS1 in HeLa cells induced exon skipping in a model β-globin pre-mRNA and a human tra-2β pre-mRNA. Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase γ-subunit pre-mRNA. The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54. RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs.
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Lallena, M. J., and I. Correas. "Transcription-dependent redistribution of nuclear protein 4.1 to SC35-enriched nuclear domains." Journal of Cell Science 110, no. 2 (January 15, 1997): 239–47. http://dx.doi.org/10.1242/jcs.110.2.239.
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Protein 4.1, originally identified as a component of the membrane-skeleton of the red blood cell, has also been localized in the nucleus of mammalian cells. To learn more about nuclear 4.1 protein, we have analyzed the nature of its association with the nuclear structure in comparison with SC35 and snRNP antigens, splicing proteins of the nuclear speckle domains. When MDCK or HeLa cells were digested with DNase I and washed in the presence of high salt (2 M NaCl), snRNP antigens were extracted whereas protein 4.1 and SC35 remained colocalizing in nuclear speckles. In cells treated with RNase A or heat shocked, nuclear 4.1 distribution also resembled that of SC35. Experiments carried out in transcriptionally active nuclei showed that protein 4.1 distributed in irregularly shaped speckles which appeared to be interconnected. During transcriptional inhibition, protein 4.1 accumulated in rounded speckles lacking interconnections. When cells were released from transcriptional inhibition, protein 4.1 redistributed back to the interconnected speckle pattern of transcriptionally active cells, as it was also observed for SC35. Finally, coprecipitation of 4.1 and SC35 proteins from RNase A digested HeLa nuclei further indicates that these two proteins are associated, forming part of the nuclear speckle domains to which they attach more tightly than snRNP antigens.
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Vottero, Alessandra, Tomoshige Kino, Herve Combe, Pierre Lecomte, and George P. Chrousos. "A Novel, C-Terminal Dominant Negative Mutation of the GR Causes Familial Glucocorticoid Resistance through Abnormal Interactions with p160 Steroid Receptor Coactivators." Journal of Clinical Endocrinology & Metabolism 87, no. 6 (June 1, 2002): 2658–67. http://dx.doi.org/10.1210/jcem.87.6.8520.
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Primary cortisol resistance is a rare, inherited or sporadic form of generalized end-organ insensitivity to glucocorticoids. Here, we report a kindred in which affected members had a heterozygous T to G base substitution at nucleotide 2373 of exon 9α of the GR gene, causing substitution of Ile by Met at position 747. This mutation was located close to helix 12, at the C terminus of the ligand-binding domain, which has a pivotal role in the formation of activation function (AF)-2, a subdomain that interacts with p160 coactivators. The affinity of the mutant GR for dexamethasone was decreased by about 2-fold, and its transcriptional activity on the glucocorticoid-responsive mouse mammary tumor virus promoter was compromised by 20- to 30-fold. In addition, the mutant GR functioned as a dominant negative inhibitor of wild-type receptor-induced transactivation. The mutant GR through its intact AF-1 domain bound to a p160 coactivator, but failed to do so through its AF-2 domain. Overexpression of a p160 coactivator restored the transcriptional activity and reversed the negative transdominant activity of the mutant GR. Interestingly, green fluorescent protein (GFP)-fused GRαI747M had a slight delay in its translocation from the cytoplasm into the nucleus and formed coarser nuclear speckles than GFP-fused wild-type GRα. Similarly, a GFP-fused p160 coactivator had a distinctly different distribution in the nucleus in the presence of mutant vs. wild-type receptor, presenting also as coarser speckling. We conclude that the mutation at amino acid 747 of the GR causes familial, autosomal dominant glucocorticoid resistance by decreasing ligand binding affinity and transcriptional activity, and by exerting a negative transdominant effect on the wild-type receptor. The mutant receptor has an ineffective AF-2 domain, which leads to an abnormal interaction with p160 coactivators and a distinct nuclear distribution of both.
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Cazalla, Demian, Kathryn Newton, and Javier F. Cáceres. "A Novel SR-Related Protein Is Required for the Second Step of Pre-mRNA Splicing." Molecular and Cellular Biology 25, no. 8 (April 15, 2005): 2969–80. http://dx.doi.org/10.1128/mcb.25.8.2969-2980.2005.
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ABSTRACT The SR family proteins and SR-related polypeptides are important regulators of pre-mRNA splicing. A novel SR-related protein of an apparent molecular mass of 53 kDa was isolated in a gene trap screen that identifies proteins which localize to the nuclear speckles. This novel protein possesses an arginine- and serine-rich domain and was termed SRrp53 (for SR-related protein of 53 kDa). In support for a role of this novel RS-containing protein in pre-mRNA splicing, we identified the mouse ortholog of the Saccharomyces cerevisiae U1 snRNP-specific protein Luc7p and the U2AF65-related factor HCC1 as interacting proteins. In addition, SRrp53 is able to interact with some members of the SR family of proteins and with U2AF35 in a yeast two-hybrid system and in cell extracts. We show that in HeLa nuclear extracts immunodepleted of SRrp53, the second step of pre-mRNA splicing is blocked, and recombinant SRrp53 is able to restore splicing activity. SRrp53 also regulates alternative splicing in a concentration-dependent manner. Taken together, these results suggest that SRrp53 is a novel SR-related protein that has a role both in constitutive and in alternative splicing.
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Sharma, Nishi R., Vladimir Majerciak, Michael J. Kruhlak, Lulu Yu, Jeong Gu Kang, Acong Yang, Shuo Gu, Marvin J. Fritzler, and Zhi-Ming Zheng. "KSHV RNA-binding protein ORF57 inhibits P-body formation to promote viral multiplication by interaction with Ago2 and GW182." Nucleic Acids Research 47, no. 17 (August 10, 2019): 9368–85. http://dx.doi.org/10.1093/nar/gkz683.
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Abstract Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.
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Edelmann, Jennifer, Tetyana Klymenko, Chen Lu, Emma Vilventhraraja, Sven Estenfelder, Eugen Tausch, Stephen A. Beers, et al. "In Chronic Lymphocytic Leukemia ΔCT7544-7545 Mutant NOTCH1 Maintains Transcription Factor Activity with Longer Lasting Effects Due to Slower Degradation." Blood 128, no. 22 (December 2, 2016): 971. http://dx.doi.org/10.1182/blood.v128.22.971.971.
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Abstract The NOTCH1 gene locusencodes a cell surface receptor which is recurrently mutated in chronic lymphocytic leukemia (CLL), with a rising incidence in higher risk cases. Upon ligand-binding two cleavage events occur. As a consequence the receptor's intracellular domain (NICD) is released and shifts to the nucleus, where it functions as a transcription factor. Since in CLL NOTCH1 mutations mainly affect the protein's PEST-domain, which is involved in NICD degradation, they were assumed to be activating mutations. However, to date this has only been proven for mutations that lead to the loss of the complete PEST-domain or major parts of it, whereas the hotspot mutation in CLL (ΔCT7544-7545; E2515fs) leads to the loss of only 39 amino acids. To prove the activating nature of the CLL hotspot mutation, we developed a cell culture based model using lentiviral transduction of B-cell lymphoma cell lines. A full-length NOTCH1 construct was cloned into a pCCL transfer vector and the CLL hotspot mutation was introduced. The cell lines SU-DHL4, Raji and Daudi were transduced with wild-type NOTCH1 (NOTCH1WT), ΔCT-mutant NOTCH1 (NOTCH1ΔCT) and empty vector. For validation experiments, primary CLL samples were screened for PEST-domain NOTCH1 mutations by targeted next-generation sequencing of exon 34. Activation of NOTCH1 was induced via treatment of cells with the Ca2+ chelator EGTA (1.0 mM, 1 hour). Liberated NICD was semi-quantitatively assessed by Western Blot and image densitometry from whole cell lysates or nuclear protein fractions. In addition, the NICD was visualized by intracellular immunofluorescent staining along with nuclear staining for DAPI and histone H3K27me / histone H3K27ac. Expression of the NOTCH1 target gene HES1 was quantified using TaqMan® RT-PCR analysis. Treatment with EGTA robustly activated NOTCH1 signalling; with the highest levels of NICD seen in the nucleus 2 to 4 hours after activation. Immunofluorescent staining of the NICD in NOTCH1 transduced cells 3 hours after activation revealed a speckled pattern in the nucleus without differences between NICDWT and NICDΔCT. Speckles co-localized with areas of relaxed chromatin, with NICD staining coincident with histone H3K27ac staining and separate from histone H3K27me staining. Co-localization of NICD speckles with areas of relaxed chromatin was confirmed in primary CLL samples where it was again observed independent of NOTCH1 mutation status. In Western Blot analyses, NICDWT and NICDΔCT were distinguishable due to their different molecular weight. Without prior activation, NOTCH1WT transduced cell lines displayed low levels of NICD. In contrast, NOTCH1ΔCT transduced cell lines presented with considerably higher levels of NICDΔCT despite comparable cell surface levels of transduced NOTCH1. After activation of NOTCH1WT and NOTCH1ΔCT with EGTA, the levels of NICDWT and NICDΔCT rose to a similar maximum, but a subsequent time-course showed a slower degradation of the NICDΔCT compared to the NICDWT. The slower degradation of mutant NICD was confirmed in primary CLL samples including samples with truncating mutations of the PEST-domain other than the hotspot mutation E2515fs (Q2440*, S2486*, Q2307*). HES1 is an established NOTCH1 target gene activated by nuclear NICD. Baseline expression levels of HES1 did not differ between NOTCH1WT and NOTCH1ΔCT transduced SU-DHL4 cells. After NOTCH1 activation, HES1 expression was up-regulated ~2.9 fold compared to baseline. Peak expression levels were seen around 3 hours after activation. HES1 expression levels fell more slowly in NOTCH1ΔCT transduced SU-DHL4 cells suggesting longer lasting effects on target genes by NICDΔCT than by NICDWT. In summary, our data demonstrates for the first time that the ΔCT7544-7545 mutant of NOTCH1 maintains transcriptional activity for longer due to slower degradation of its NICD. Our understanding about NICD target genes remains incomplete, but their identification will be crucial to understand the role of NOTCH1 mutations in CLL pathogenesis. Disclosures Tausch: Gilead: Other: Travel support, Speakers Bureau; Celgene: Other: Travel support; Amgen: Other: Travel support. Stilgenbauer:Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding. Cragg:Roche: Consultancy, Research Funding; GSK: Research Funding; Bioinvent International: Consultancy, Research Funding; Baxalta: Consultancy; Gilead Sciences: Research Funding.
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Papoutsopoulou, Stamatia, and Ralf Janknecht. "Phosphorylation of ETS Transcription Factor ER81 in a Complex with Its Coactivators CREB-Binding Protein and p300." Molecular and Cellular Biology 20, no. 19 (October 1, 2000): 7300–7310. http://dx.doi.org/10.1128/mcb.20.19.7300-7310.2000.
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ABSTRACT The ETS protein ER81 is a DNA-binding factor capable of enhancing gene transcription and is implicated in cellular transformation, but presently the mechanisms of its actions are unclear. In this report, ER81 is shown to coimmunoprecipitate with the transcriptional coactivator CREB-binding protein (CBP) and the related p300 protein (together referred to as CBP/p300). Moreover, confocal laser microscopic studies demonstrated that ER81 and p300 colocalized to nuclear speckles. In vitro and in vivo interaction studies revealed that ER81 amino acids 249 to 429, which encompass the ETS DNA-binding domain, are responsible for binding to CBP/p300. However, mutation of a putative protein-protein interaction motif, LXXLL, in the ETS domain of ER81 did not affect interaction with CBP/p300, whereas DNA binding of ER81 was abolished. Furthermore, two regions within CBP, amino acids 451 to 721 and 1891 to 2175, are capable of binding to ER81. Consistent with the physical interaction between ER81 and the coactivators CBP and p300, ER81 transcriptional activity was potentiated by CBP/p300 overexpression. Moreover, an ER81-associated protein kinase activity was enhanced upon p300 overexpression. This protein kinase phosphorylates ER81 on serines 191 and 216, and mutation of these phosphorylation sites increased ER81 transcriptional activity in Mv1Lu cells but not in HeLa cells. Altogether, our data elucidate the mechanism of how ER81 regulates gene transcription, through interaction with the coactivators CBP and p300 and an associated kinase that may cell type specifically modulate the ability of ER81 to activate gene transcription.
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Melčák, Ivo, Štěpánka Melčáková, Vojtěch Kopsky, Jaromı́ra Večeřová, and Ivan Raška. "Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles." Molecular Biology of the Cell 12, no. 2 (February 2001): 393–406. http://dx.doi.org/10.1091/mbc.12.2.393.
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Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed.
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Boronenkov, Igor V., Joost C. Loijens, Masato Umeda, and Richard A. Anderson. "Phosphoinositide Signaling Pathways in Nuclei Are Associated with Nuclear Speckles Containing Pre-mRNA Processing Factors." Molecular Biology of the Cell 9, no. 12 (December 1998): 3547–60. http://dx.doi.org/10.1091/mbc.9.12.3547.
Full textAbstract:
Phosphoinositide signal transduction pathways in nuclei use enzymes that are indistinguishable from their cytosolic analogues. We demonstrate that distinct phosphatidylinositol phosphate kinases (PIPKs), the type I and type II isoforms, are concentrated in nuclei of mammalian cells. The cytosolic and nuclear PIPKs display comparable activities toward the substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate. Indirect immunofluorescence revealed that these kinases were associated with distinct subnuclear domains, identified as “nuclear speckles,” which also contained pre-mRNA processing factors. A pool of nuclear phosphatidylinositol bisphosphate (PIP2), the product of these kinases, was also detected at these same sites by monoclonal antibody staining. The localization of PIPKs and PIP2 to speckles is dynamic in that both PIPKs and PIP2 reorganize along with other speckle components upon inhibition of mRNA transcription. Because PIPKs have roles in the production of most phosphatidylinositol second messengers, these findings demonstrate that phosphatidylinositol signaling pathways are localized at nuclear speckles. Surprisingly, the PIPKs and PIP2 are not associated with invaginations of the nuclear envelope or any nuclear membrane structure. The putative absence of membranes at these sites suggests novel mechanisms for the generation of phosphoinositides within these structures.
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Chen, Yu, Yang Zhang, Yuchuan Wang, Liguo Zhang, Eva K. Brinkman, Stephen A. Adam, Robert Goldman, Bas van Steensel, Jian Ma, and Andrew S. Belmont. "Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler." Journal of Cell Biology 217, no. 11 (August 28, 2018): 4025–48. http://dx.doi.org/10.1083/jcb.201807108.
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While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a “cytological ruler” for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription “hot zones.” Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression.
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Singh, Parmit Kumar, Gregory J. Bedwell, and Alan N. Engelman. "Spatial and Genomic Correlates of HIV-1 Integration Site Targeting." Cells 11, no. 4 (February 14, 2022): 655. http://dx.doi.org/10.3390/cells11040655.
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HIV-1 integrase and capsid proteins interact with host proteins to direct preintegration complexes to active transcription units within gene-dense regions of chromosomes for viral DNA integration. Analyses of spatially-derived genomic DNA coordinates, such as nuclear speckle-associated domains, lamina-associated domains, super enhancers, and Spatial Position Inference of the Nuclear (SPIN) genome states, have further informed the mechanisms of HIV-1 integration targeting. Critically, however, these different types of genomic coordinates have not been systematically analyzed to synthesize a concise description of the regions of chromatin that HIV-1 prefers for integration. To address this informational gap, we have extensively correlated genomic DNA coordinates of HIV-1 integration targeting preferences. We demonstrate that nuclear speckle-associated and speckle-proximal chromatin are highly predictive markers of integration and that these regions account for known HIV biases for gene-dense regions, highly transcribed genes, as well as the mid-regions of gene bodies. In contrast to a prior report that intronless genes were poorly targeted for integration, we find that intronless genes in proximity to nuclear speckles are more highly targeted than are spatially-matched intron-containing genes. Our results additionally highlight the contributions of capsid and integrase interactions with respective CPSF6 and LEDGF/p75 host factors in these HIV-1 integration targeting preferences.
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Hu, Yan, Igor Kireev, Matt Plutz, Nazanin Ashourian, and Andrew S. Belmont. "Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template." Journal of Cell Biology 185, no. 1 (April 6, 2009): 87–100. http://dx.doi.org/10.1083/jcb.200809196.
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The structure of interphase chromosomes, and in particular the changes in large-scale chromatin structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1–2 Mbp chromatin domains formed by multi-copy BAC transgenes containing 130–220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale chromatin fibers compacted nonuniformly well above the 30-nm chromatin fiber. An approximately 1.5–3-fold extension of these large-scale chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock–induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization.
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Bregman, D. B., L. Du, S. van der Zee, and S. L. Warren. "Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains." Journal of Cell Biology 129, no. 2 (April 15, 1995): 287–98. http://dx.doi.org/10.1083/jcb.129.2.287.
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A subpopulation of the largest subunit of RNA polymerase II (Pol II LS) is located in 20-50 discrete subnuclear domains that are closely linked to speckle domains, which store splicing proteins. The speckle-associated fraction of Pol II LS is hyperphosphorylated on the COOH-terminal domain (CTD), and it is highly resistant to extraction by detergents. A diffuse nucleoplasmic fraction of Pol II LS is relatively hypophosphorylated on the CTD, and it is easily extracted by detergents. In transcriptionally active nuclei, speckle bound hyperphosphorylated Pol II LS molecules are distributed in irregularly shaped speckle domains, which appear to be interconnected via a reticular network. When transcription is inhibited, hyperphosphorylated Pol II LS and splicing protein SC35 accumulate in speckle domains, which are transformed into enlarged, dot-like structures lacking interconnections. When cells are released from transcriptional inhibition, Pol IIO and SC35 redistribute back to the interconnected speckle pattern of transcriptionally active cells. The redistribution of Pol II and SC35 is synchronous, reversible, and temperature dependent. It is concluded that: (a) hyperphosphorylation of Pol II LS's CTD is a better indicator of its tight association to discrete subnuclear domains than its transcriptional activity; (b) during states of transcriptional inhibition, hyperphosphorylated Pol II LS can be stored in enlarged speckle domains, which under the light microscope appear to coincide with the storage sites for splicing proteins; and (c) Pol II and splicing proteins redistribute simultaneously according to the overall transcriptional activity of the nucleus.