Articles de revues : « Cotranscriptionality »

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Aslanzadeh, Vahid, Yuanhua Huang, Guido Sanguinetti et Jean D. Beggs. « Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast ». Genome Research 28, n^o 2 (18 décembre 2017) : 203–13. http://dx.doi.org/10.1101/gr.225615.117.

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Perales, Roberto, et David Bentley. « “Cotranscriptionality” : The Transcription Elongation Complex as a Nexus for Nuclear Transactions ». Molecular Cell 36, n^o 2 (octobre 2009) : 178–91. http://dx.doi.org/10.1016/j.molcel.2009.09.018.

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Aslanzadeh, Vahid, Yuanhua Huang, Guido Sanguinetti et Jean D. Beggs. « Corrigendum : Transcription rate strongly affects splicing fidelity and cotranscriptionality in budding yeast ». Genome Research 28, n^o 4 (avril 2018) : 606.2. http://dx.doi.org/10.1101/gr.236265.118.

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Koš, Martin, et David Tollervey. « Yeast Pre-rRNA Processing and Modification Occur Cotranscriptionally ». Molecular Cell 37, n^o 6 (mars 2010) : 809–20. http://dx.doi.org/10.1016/j.molcel.2010.02.024.

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Nawroth, I., F. Mueller, E. Basyuk, N. Beerens, U. L. Rahbek, X. Darzacq, E. Bertrand, J. Kjems et U. Schmidt. « Stable assembly of HIV-1 export complexes occurs cotranscriptionally ». RNA 20, n^o 1 (19 novembre 2013) : 1–8. http://dx.doi.org/10.1261/rna.038182.113.

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Pendleton, Kathryn E., Sung-Kyun Park, Olga V. Hunter, Stefan M. Bresson et Nicholas K. Conrad. « Balance between MAT2A intron detention and splicing is determined cotranscriptionally ». RNA 24, n^o 6 (21 mars 2018) : 778–86. http://dx.doi.org/10.1261/rna.064899.117.

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Li, Jiang, Jie Chao, Jiye Shi et Chunhai Fan. « Cotranscriptionally Folded RNA Nanostructures Pave the Way to Intracellular Nanofabrication ». ChemBioChem 16, n^o 1 (21 novembre 2014) : 39–41. http://dx.doi.org/10.1002/cbic.201402627.

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Schmidt, Ute, Eugenia Basyuk, Marie-Cécile Robert, Minoru Yoshida, Jean-Philippe Villemin, Didier Auboeuf, Stuart Aitken et Edouard Bertrand. « Real-time imaging of cotranscriptional splicing reveals a kinetic model that reduces noise : implications for alternative splicing regulation ». Journal of Cell Biology 193, n^o 5 (30 mai 2011) : 819–29. http://dx.doi.org/10.1083/jcb.201009012.

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Splicing is a key process that expands the coding capacity of genomes. Its kinetics remain poorly characterized, and the distribution of splicing time caused by the stochasticity of single splicing events is expected to affect regulation efficiency. We conducted a small-scale survey on 40 introns in human cells and observed that most were spliced cotranscriptionally. Consequently, we constructed a reporter system that splices cotranscriptionally and can be monitored in live cells and in real time through the use of MS2–GFP. All small nuclear ribonucleoproteins (snRNPs) are loaded on nascent pre-mRNAs, and spliceostatin A inhibits splicing but not snRNP recruitment. Intron removal occurs in minutes and is best described by a model where several successive steps are rate limiting. Each pre-mRNA molecule is predicted to require a similar time to splice, reducing kinetic noise and improving the regulation of alternative splicing. This model is relevant to other kinetically controlled processes acting on few molecules.

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Wuarin, J., et U. Schibler. « Physical isolation of nascent RNA chains transcribed by RNA polymerase II : evidence for cotranscriptional splicing ». Molecular and Cellular Biology 14, n^o 11 (novembre 1994) : 7219–25. http://dx.doi.org/10.1128/mcb.14.11.7219-7225.1994.

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In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.

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Wuarin, J., et U. Schibler. « Physical isolation of nascent RNA chains transcribed by RNA polymerase II : evidence for cotranscriptional splicing. » Molecular and Cellular Biology 14, n^o 11 (novembre 1994) : 7219–25. http://dx.doi.org/10.1128/mcb.14.11.7219.

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In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.

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Trcek, T., et R. H. Singer. « The cytoplasmic fate of an mRNP is determined cotranscriptionally : exception or rule ? » Genes & ; Development 24, n^o 17 (1 septembre 2010) : 1827–31. http://dx.doi.org/10.1101/gad.1972810.

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Dye, Michael J., et Nick J. Proudfoot. « Terminal Exon Definition Occurs Cotranscriptionally and Promotes Termination of RNA Polymerase II ». Molecular Cell 3, n^o 3 (mars 1999) : 371–78. http://dx.doi.org/10.1016/s1097-2765(00)80464-5.

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Huertas, Pablo, et Andrés Aguilera. « Cotranscriptionally Formed DNA:RNA Hybrids Mediate Transcription Elongation Impairment and Transcription-Associated Recombination ». Molecular Cell 12, n^o 3 (septembre 2003) : 711–21. http://dx.doi.org/10.1016/j.molcel.2003.08.010.

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Li, Chen, Caryn S. Gonsalves, Marthe-Sandrine Eiymo Mwa Mpollo, Punam Malik, Stanley M. Tahara et Vijay K. Kalra. « MicroRNA 648 Targets ET-1 mRNA and Is Cotranscriptionally Regulated withMICAL3by PAX5 ». Molecular and Cellular Biology 35, n^o 3 (17 novembre 2014) : 514–28. http://dx.doi.org/10.1128/mcb.01199-14.

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Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene,MICAL3(microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3′ untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3′ UTR of ET-1 mRNA. Since miR-648 is located in a 5′-proximal intron ofMICAL3, we examined which of three potential promoters was responsible for its expression. TheMICAL3distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.

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Eckmann, Christian R., et Michael F. Jantsch. « The RNA-editing Enzyme ADAR1 Is Localized to the Nascent Ribonucleoprotein Matrix on Xenopus Lampbrush Chromosomes but Specifically Associates with an Atypical Loop ». Journal of Cell Biology 144, n^o 4 (22 février 1999) : 603–15. http://dx.doi.org/10.1083/jcb.144.4.603.

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Double-stranded RNA adenosine deaminase (ADAR1, dsRAD, DRADA) converts adenosines to inosines in double-stranded RNAs. Few candidate substrates for ADAR1 editing are known at this point and it is not known how substrate recognition is achieved. In some cases editing sites are defined by basepaired regions formed between intronic and exonic sequences, suggesting that the enzyme might function cotranscriptionally. We have isolated two variants of Xenopus laevis ADAR1 for which no editing substrates are currently known. We demonstrate that both variants of the enzyme are associated with transcriptionally active chromosome loops suggesting that the enzyme acts cotranscriptionally. The widespread distribution of the protein along the entire chromosome indicates that ADAR1 associates with the RNP matrix in a substrate-independent manner. Inhibition of splicing, another cotranscriptional process, does not affect the chromosomal localization of ADAR1. Furthermore, we can show that the enzyme is dramatically enriched on a special RNA-containing loop that seems transcriptionally silent. Detailed analysis of this loop suggests that it might represent a site of ADAR1 storage or a site where active RNA editing is taking place. Finally, mutational analysis of ADAR1 demonstrates that a putative Z-DNA binding domain present in ADAR1 is not required for chromosomal targeting of the protein.

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West, Steven, Natalia Gromak, Christopher J. Norbury et Nicholas J. Proudfoot. « Adenylation and Exosome-Mediated Degradation of Cotranscriptionally Cleaved Pre-Messenger RNA in Human Cells ». Molecular Cell 21, n^o 3 (février 2006) : 437–43. http://dx.doi.org/10.1016/j.molcel.2005.12.008.

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Tennyson, Christine N., Henry J. Klamut et Ronald G. Worton. « The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced ». Nature Genetics 9, n^o 2 (février 1995) : 184–90. http://dx.doi.org/10.1038/ng0295-184.

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Wery, M., S. Ruidant, S. Schillewaert, N. Lepore et D. L. J. Lafontaine. « The nuclear poly(A) polymerase and Exosome cofactor Trf5 is recruited cotranscriptionally to nucleolar surveillance ». RNA 15, n^o 3 (20 janvier 2009) : 406–19. http://dx.doi.org/10.1261/rna.1402709.

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Hessle, Viktoria, Petra Björk, Marcus Sokolowski, Ernesto González de Valdivia, Rebecca Silverstein, Konstantin Artemenko, Anu Tyagi et al. « The Exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins ». Molecular Biology of the Cell 20, n^o 15 (août 2009) : 3459–70. http://dx.doi.org/10.1091/mbc.e09-01-0079.

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Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins.

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Walters, Robert W., Tyler Matheny, Laura S. Mizoue, Bhalchandra S. Rao, Denise Muhlrad et Roy Parker. « Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae ». Proceedings of the National Academy of Sciences 114, n^o 3 (28 décembre 2016) : 480–85. http://dx.doi.org/10.1073/pnas.1619369114.

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RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5′ nicotinamide-adenine dinucleotide (NAD+) RNA in bacteria. Whether 5′ NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5′ NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae. NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5′ end processed. These results define an additional 5′ RNA cap structure in eukaryotes and raise the possibility that this 5′ NAD+ cap could modulate RNA stability and translation on specific subclasses of mRNAs.

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Wang, Jinkai. « Integrative analyses of transcriptome data reveal the mechanisms of post-transcriptional regulation ». Briefings in Functional Genomics 20, n^o 4 (22 février 2021) : 207–12. http://dx.doi.org/10.1093/bfgp/elab004.

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Abstract Post-transcriptional processing of RNAs plays important roles in a variety of physiological and pathological processes. These processes can be precisely controlled by a series of RNA binding proteins and cotranscriptionally regulated by transcription factors as well as histone modifications. With the rapid development of high-throughput sequencing techniques, multiomics data have been broadly used to study the mechanisms underlying the important biological processes. However, how to use these high-throughput sequencing data to elucidate the fundamental regulatory roles of post-transcriptional processes is still of great challenge. This review summarizes the regulatory mechanisms of post-transcriptional processes and the general principles and approaches to dissect these mechanisms by integrating multiomics data as well as public resources.

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Han, Yo-Sub, Hwee Kim, Trent A. Rogers et Shinnosuke Seki. « Self-Attraction Removal from Oritatami Systems ». International Journal of Foundations of Computer Science 30, n^o 06n07 (septembre 2019) : 1047–67. http://dx.doi.org/10.1142/s0129054119400288.

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RNA cotranscriptional folding refers to the phenomenon in which an RNA transcript folds upon itself while being synthesized out of a gene by an RNA polymerase. Oritatami is a computational model of this phenomenon, which lets its sequence of beads (abstract molecules) taken from a finite alphabet [Formula: see text] fold cotranscriptionally via interactions between beads according to its rule set. In this paper, we study the problem of removing self-attractions, which lets a bead interact with another bead of the same kind, from a given oritatami system without changing its behavior. Self-attraction is one of the major challenges in the construction of intrinsic oritatami systems, which can simulate even the dynamics of all the oritatami systems.

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Ninomiya, Kensuke, Naoyuki Kataoka et Masatoshi Hagiwara. « Stress-responsive maturation of Clk1/4 pre-mRNAs promotes phosphorylation of SR splicing factor ». Journal of Cell Biology 195, n^o 1 (26 septembre 2011) : 27–40. http://dx.doi.org/10.1083/jcb.201107093.

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It has been assumed that premessenger ribonucleic acids (RNAs; pre-mRNAs) are spliced cotranscriptionally in the process of gene expression. However, in this paper, we report that splicing of Clk1/4 mRNAs is suspended in tissues and cultured cells and that intermediate forms retaining specific introns are abundantly pooled in the nucleus. Administration of the Cdc2-like kinase–specific inhibitor TG003 increased the level of Clk1/4 mature mRNAs by promoting splicing of the intron-retaining RNAs. Under stress conditions, splicing of general pre-mRNAs was inhibited by dephosphorylation of SR splicing factors, but exposure to stresses, such as heat shock and osmotic stress, promoted the maturation of Clk1/4 mRNAs. Clk1/4 proteins translated after heat shock catalyzed rephosphorylation of SR proteins, especially SRSF4 and SRSF10. These findings suggest that Clk1/4 expression induced by stress-responsive splicing serves to maintain the phosphorylation state of SR proteins.

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Chen, Juan, Zhaokui Cai, Meizhu Bai, Xiaohua Yu, Chao Zhang, Changchang Cao, Xihao Hu et al. « The RNA-binding protein ROD1/PTBP3 cotranscriptionally defines AID-loading sites to mediate antibody class switch in mammalian genomes ». Cell Research 28, n^o 10 (24 août 2018) : 981–95. http://dx.doi.org/10.1038/s41422-018-0076-9.

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Visa, N., E. Izaurralde, J. Ferreira, B. Daneholt et I. W. Mattaj. « A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. » Journal of Cell Biology 133, n^o 1 (1 avril 1996) : 5–14. http://dx.doi.org/10.1083/jcb.133.1.5.

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In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.

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Wu, Chenglei, Weixin Chen, Jincan He, Shouheng Jin, Yukun Liu, Yang Yi, Zhuoxing Gao et al. « Interplay of m6A and H3K27 trimethylation restrains inflammation during bacterial infection ». Science Advances 6, n^o 34 (août 2020) : eaba0647. http://dx.doi.org/10.1126/sciadv.aba0647.

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While N6-methyladenosine (m6A) is the most prevalent modification of eukaryotic messenger RNA (mRNA) involved in various cellular responses, its role in modulating bacteria-induced inflammatory response remains elusive. Here, we showed that loss of the m6A reader YTH-domain family 2 (YTHDF2) promoted demethylation of histone H3 lysine-27 trimethylation (H3K27me3), which led to enhanced production of proinflammatory cytokines and facilitated the deposition of m6A cotranscriptionally. Mechanistically, the mRNA of lysine demethylase 6B (KDM6B) was m6A-modified and its decay mediated by YTHDF2. YTHDF2 deficiency stabilized KDM6B to promote H3K27me3 demethylation of multiple proinflammatory cytokines and subsequently enhanced their transcription. Furthermore, we identified H3K27me3 as a barrier for m6A modification during transcription. KDM6B recruits the m6A methyltransferase complex to facilitate the methylation of m6A in transcribing mRNA by removing adjacent H3K27me3 barriers. These results revealed cross-talk between m6A and H3K27me3 during bacterial infection, which has broader implications for deciphering epitranscriptomics in immune homeostasis.

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Kiesler, Eva, Manuela E. Hase, David Brodin et Neus Visa. « Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs ». Journal of Cell Biology 168, n^o 7 (21 mars 2005) : 1013–25. http://dx.doi.org/10.1083/jcb.200407173.

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Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59–RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation.

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Björk, Petra, Jan-Olov Persson et Lars Wieslander. « Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo ». Journal of Cell Biology 211, n^o 1 (12 octobre 2015) : 63–75. http://dx.doi.org/10.1083/jcb.201412017.

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Eukaryotic gene expression requires the ordered association of numerous factors with precursor messenger RNAs (premRNAs)/messenger RNAs (mRNAs) to achieve efficiency and regulation. Here, we use the Balbiani ring (BR) genes to demonstrate the temporal and spatial association of the exon junction complex (EJC) core with gene-specific endogenous premRNAs and mRNAs. The EJC core components bind cotranscriptionally to BR premRNAs during or very rapidly after splicing. The EJC core does not recruit the nonsense-mediated decay mediaters UPF2 and UPF3 until the BR messenger RNA protein complexes (mRNPs) enter the interchromatin. Even though several known adapters for the export factor NXF1 become part of BR mRNPs already at the gene, NXF1 binds to BR mRNPs only in the interchromatin. In steady state, a subset of the BR mRNPs in the interchromatin binds NXF1, UPF2, and UPF3. This binding appears to occur stochastically, and the efficiency approximately equals synthesis and export of the BR mRNPs. Our data provide unique in vivo information on how export competent eukaryotic mRNPs are formed.

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Prather, Donald, Nevan J. Krogan, Andrew Emili, Jack F. Greenblatt et Fred Winston. « Identification and Characterization of Elf1, a Conserved Transcription Elongation Factor in Saccharomyces cerevisiae ». Molecular and Cellular Biology 25, n^o 22 (15 novembre 2005) : 10122–35. http://dx.doi.org/10.1128/mcb.25.22.10122-10135.2005.

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ABSTRACT In order to identify previously unknown transcription elongation factors, a genetic screen was carried out to identify mutations that cause lethality when combined with mutations in the genes encoding the elongation factors TFIIS and Spt6. This screen identified a mutation in YKL160W, hereafter named ELF1 (elongation factor 1). Further analysis identified synthetic lethality between an elf1Δ mutation and mutations in genes encoding several known elongation factors, including Spt4, Spt5, Spt6, and members of the Paf1 complex. Genome-wide synthetic lethality studies confirmed that elf1Δ specifically interacts with mutations in genes affecting transcription elongation. Chromatin immunoprecipitation experiments show that Elf1 is cotranscriptionally recruited over actively transcribed regions and that this association is partially dependent on Spt4 and Spt6. Analysis of elf1Δ mutants suggests a role for this factor in maintaining proper chromatin structure in regions of active transcription. Finally, purification of Elf1 suggests an association with casein kinase II, previously implicated in roles in transcription. Together, these results suggest an important role for Elf1 in the regulation of transcription elongation.

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Malagon, Francisco, et Torben Heick Jensen. « The T Body, a New Cytoplasmic RNA Granule in Saccharomyces cerevisiae ». Molecular and Cellular Biology 28, n^o 19 (4 août 2008) : 6022–32. http://dx.doi.org/10.1128/mcb.00684-08.

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ABSTRACT A large share of mRNA processing and packaging events occurs cotranscriptionally. To explore the hypothesis that transcription defects may affect mRNA fate, we analyzed poly(A)+ RNA distribution in Saccharomyces cerevisiae strains harboring mutations in Rpb1p, the largest subunit of RNA polymerase II. In certain rpb1 mutants, a poly(A)+ RNA granule, distinct from any known structure, strongly accumulated in a confined space of the cytoplasm. RNA and protein expressed from Ty1 retrovirus-like elements colocalized with this new granule, which we have consequently named the T body. A visual screen revealed that the deletion of most genes with proposed functions in Ty1 biology unexpectedly does not alter T-body levels. In contrast, the deletion of genes encoding the Mediator transcription initiation factor subunits Srb2p and Srb5p as well as the Ty1 transcriptional regulator Spt21p greatly enhances T-body formation. Our data disclose a new cellular body putatively involved in the assembly of Ty1 particles and suggest that the cytoplasmic fate of mRNA can be affected by transcription initiation events.

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Ginsburg, Daniel S., Chhabi K. Govind et Alan G. Hinnebusch. « NuA4 Lysine Acetyltransferase Esa1 Is Targeted to Coding Regions and Stimulates Transcription Elongation with Gcn5 ». Molecular and Cellular Biology 29, n^o 24 (12 octobre 2009) : 6473–87. http://dx.doi.org/10.1128/mcb.01033-09.

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ABSTRACT NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

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Percipalle, Piergiorgio, Jian Zhao, Brian Pope, Alan Weeds, Uno Lindberg et Bertil Daneholt. « Actin Bound to the Heterogeneous Nuclear Ribonucleoprotein Hrp36 Is Associated with Balbiani Ring mRNA from the Gene to Polysomes ». Journal of Cell Biology 153, n^o 1 (2 avril 2001) : 229–36. http://dx.doi.org/10.1083/jcb.153.1.229.

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In the salivary glands of the dipteran Chironomus tentans, a specific messenger ribonucleoprotein (mRNP) particle, the Balbiani ring (BR) granule, can be visualized during its assembly on the gene and during its nucleocytoplasmic transport. We now show with immunoelectron microscopy that actin becomes associated with the BR particle concomitantly with transcription and is present in the particle in the nucleoplasm. DNase I affinity chromatography experiments with extracts from tissue culture cells indicate that both nuclear and cytoplasmic actin are bound to the heterogeneous RNP (hnRNP) protein hrp36, but not to the hnRNP proteins hrp23 and hrp45. The interaction is likely to be direct as purified actin binds to recombinant hrp36 in vitro. Furthermore, it is demonstrated by cross linking that nuclear as well as cytoplasmic actin are bound to hrp36 in vivo. It is known that hrp36 is added cotranscriptionally along the BR mRNA molecule and accompanies the RNA through the nuclear pores and into polysomes. We conclude that actin is likely to be bound to the BR transcript via hrp36 during the transfer of the mRNA from the gene all the way into polysomes.

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Kim, Geon-Woo, et Aleem Siddiqui. « Hepatitis B virus X protein recruits methyltransferases to affect cotranscriptional N6-methyladenosine modification of viral/host RNAs ». Proceedings of the National Academy of Sciences 118, n^o 3 (4 janvier 2021) : e2019455118. http://dx.doi.org/10.1073/pnas.2019455118.

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Chronic hepatitis B virus (HBV) infections are one of the leading causes of cirrhosis and hepatocellular carcinoma. N6-methyladenosine (m6A) modification of cellular and viral RNAs is the most prevalent internal modification that occurs cotranscriptionally. Previously, we reported the dual functional role of m6A modification of HBV transcripts in the viral life cycle. Here, we show that viral HBV X (HBx) protein is responsible for the m6A modifications of viral transcripts. HBV genomes defective in HBx failed to induce m6A modifications of HBV RNAs during infection/transfection, while ectopic expression of HBx restores m6A modifications of the viral RNAs but not the mutant HBx carrying the nuclear export signal. Using chromatin immunoprecipitation assays, we provide evidence that HBx and m6A methyltransferase complexes are localized on the HBV minichromosome to achieve cotranscriptional m6A modification of viral RNAs. HBx interacts with METTL3 and 14 to carry out methylation activity and also modestly stimulates their nuclear import. This role of HBx in mediating m6A modification also extends to host phosphatase and tensin homolog (PTEN) mRNA. This study provides insight into how a viral protein recruits RNA methylation machinery to m6A-modify RNAs.

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Cardinale, Stefano, Barbara Cisterna, Paolo Bonetti, Chiara Aringhieri, Marco Biggiogera et Silvia M. L. Barabino. « Subnuclear Localization and Dynamics of the Pre-mRNA 3′ End Processing Factor Mammalian Cleavage Factor I 68-kDa Subunit ». Molecular Biology of the Cell 18, n^o 4 (avril 2007) : 1282–92. http://dx.doi.org/10.1091/mbc.e06-09-0846.

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Mammalian cleavage factor I (CF Im) is an essential factor that is required for the first step in pre-mRNA 3′ end processing. Here, we characterize CF Im68 subnuclear distribution and mobility. Fluorescence microscopy reveals that in addition to paraspeckles CF Im68 accumulates in structures that partially overlap with nuclear speckles. Analysis of synchronized cells shows that CF Im68 distribution in speckles and paraspeckles varies during the cell cycle. At an ultrastructural level, CF Im68 is associated with perichromatin fibrils, the sites of active transcription, and concentrates in interchromatin granules-associated zones. We show that CFIm68 colocalizes with bromouridine, RNA polymerase II, and the splicing factor SC35. On inhibition of transcription, endogenous CF Im68 no longer associates with perichromatin fibrils, but it can still be detected in interchromatin granules-associated zones. These observations support the idea that not only splicing but also 3′ end processing occurs cotranscriptionally. Finally, fluorescence recovery after photobleaching analysis reveals that the CF Im68 fraction associated with paraspeckles moves at a rate similar to the more dispersed molecules in the nucleoplasm, demonstrating the dynamic nature of this compartment. These findings suggest that paraspeckles are a functional compartment involved in RNA metabolism in the cell nucleus.

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Fincher, Justin A., Gary S. Tyson et Jonathan H. Dennis. « DNA-Encoded Chromatin Structural Intron Boundary Signals Identify Conserved Genes with Common Function ». International Journal of Genomics 2015 (2015) : 1–10. http://dx.doi.org/10.1155/2015/167578.

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The regulation of metazoan gene expression occurs in part by pre-mRNA splicing into mature RNAs. Signals affecting the efficiency and specificity with which introns are removed have not been completely elucidated. Splicing likely occurs cotranscriptionally, with chromatin structure playing a key regulatory role. We calculated DNA encoded nucleosome occupancy likelihood (NOL) scores at the boundaries between introns and exons across five metazoan species. We found that (i) NOL scores reveal a sequence-based feature at the introns on both sides of the intron-exon boundary; (ii) this feature is not part of any recognizable consensus sequence; (iii) this feature is conserved throughout metazoa; (iv) this feature is enriched in genes sharing similar functions: ATPase activity, ATP binding, helicase activity, and motor activity; (v) genes with these functions exhibit different genomic characteristics; (vi)in vivonucleosome positioning data confirm ontological enrichment at this feature; and (vii) genes with this feature exhibit unique dinucleotide distributions at the intron-exon boundary. The NOL scores point toward a physical property of DNA that may play a role in the mechanism of pre-mRNA splicing. These results provide a foundation for identification of a new set of regulatory DNA elements involved in splicing regulation.

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Byrne, Elaine M., et Jonatha M. Gott. « Unexpectedly Complex Editing Patterns at Dinucleotide Insertion Sites in Physarum Mitochondria ». Molecular and Cellular Biology 24, n^o 18 (15 septembre 2004) : 7821–28. http://dx.doi.org/10.1128/mcb.24.18.7821-7828.2004.

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ABSTRACT Many of the RNAs transcribed from the mitochondrial genome of Physarum polycephalum are edited by the insertion of nonencoded nucleotides, which are added either singly or as dinucleotides. In addition, at least one mRNA is also subject to substitutional editing in which encoded C residues are changed to U residues posttranscriptionally. We have shown previously that the predominant type of editing in these organelles, the insertion of nonencoded single C residues, occurs cotranscriptionally at the growing end of the RNA chain. However, less is known about the timing of dinucleotide addition, and it has been suggested that these insertions occur at a later stage in RNA maturation. Here we examine the addition of both single nucleotides and dinucleotides into nascent RNAs synthesized in vitro and in vivo. The distribution of added nucleotides within individual cloned cDNAs supports the hypothesis that all insertion sites are processed at the same time relative to transcription. In addition, the patterns of partial editing and misediting observed within these nascent RNAs suggest that separate factors may be required at a subset of dinucleotide insertion sites and raise the possibility that in vivo, nucleotides may be added to RNA and then changed posttranscriptionally.

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Doyle, Michael, et Michael F. Jantsch. « Distinct in vivo roles for double-stranded RNA-binding domains of the Xenopus RNA-editing enzyme ADAR1 in chromosomal targeting ». Journal of Cell Biology 161, n^o 2 (28 avril 2003) : 309–19. http://dx.doi.org/10.1083/jcb.200301034.

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The RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) deaminates adenosines to inosines in double-stranded RNA substrates. Currently, it is not clear how the enzyme targets and discriminates different substrates in vivo. However, it has been shown that the deaminase domain plays an important role in distinguishing various adenosines within a given substrate RNA in vitro. Previously, we could show that Xenopus ADAR1 is associated with nascent transcripts on transcriptionally active lampbrush chromosomes, indicating that initial substrate binding and possibly editing itself occurs cotranscriptionally. Here, we demonstrate that chromosomal association depends solely on the three double-stranded RNA-binding domains (dsRBDs) found in the central part of ADAR1, but not on the Z-DNA–binding domain in the NH2 terminus nor the catalytic deaminase domain in the COOH terminus of the protein. Most importantly, we show that individual dsRBDs are capable of recognizing different chromosomal sites in an apparently specific manner. Thus, our results not only prove the requirement of dsRBDs for chromosomal targeting, but also show that individual dsRBDs have distinct in vivo localization capabilities that may be important for initial substrate recognition and subsequent editing specificity.

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Gnan, Stefano, Mélody Matelot, Marion Weiman, Olivier Arnaiz, Frédéric Guérin, Linda Sperling, Mireille Bétermier, Claude Thermes, Chun-Long Chen et Sandra Duharcourt. « GC content, but not nucleosome positioning, directly contributes to intron splicing efficiency in Paramecium ». Genome Research 32, n^o 4 (9 mars 2022) : 699–709. http://dx.doi.org/10.1101/gr.276125.121.

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Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the more than 90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur cotranscriptionally, and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We show that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of ∼151 bp, among the smallest periodicities reported. Our analysis has revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint favoring introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for nonsense-mediated decay effectors, we show that introns located at the edge of nucleosomes display higher splicing efficiency than those at the center. However, multiple regression analysis indicates that the low GC content of introns, rather than nucleosome positioning, is associated with high splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning, and intron evolution in Paramecium.

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Kotovic, Kimberly M., Daniel Lockshon, Lamia Boric et Karla M. Neugebauer. « Cotranscriptional Recruitment of the U1 snRNP to Intron-Containing Genes in Yeast ». Molecular and Cellular Biology 23, n^o 16 (15 août 2003) : 5768–79. http://dx.doi.org/10.1128/mcb.23.16.5768-5779.2003.

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ABSTRACT Evidence that pre-mRNA processing events are temporally and, in some cases, mechanistically coupled to transcription has led to the proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. Here we address two key questions raised by this proposal: (i) whether the U1 snRNP, which binds to the 5′ splice site of each intron, is recruited cotranscriptionally in vivo and, (ii) if so, where along the length of active genes the U1 snRNP is concentrated. Using chromatin immunoprecipitation (ChIP) in yeast, we show that elevated levels of the U1 snRNP were specifically detected in gene regions containing introns and downstream of introns but not along the length of intronless genes. In contrast to capping enzymes, which bind directly to Pol II, the U1 snRNP was poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP was dependent on RNA synthesis and was abolished by intron removal. Microarray analysis revealed that intron-containing genes were preferentially selected by ChIP with the U1 snRNP. Thus, U1 snRNP accumulation at genes correlated with the presence and position of introns, indicating that introns are necessary for cotranscriptional U1 snRNP recruitment and/or retention.

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Smith, Kelly P., Phillip T. Moen, Karen L. Wydner, John R. Coleman et Jeanne B. Lawrence. « Processing of Endogenous Pre-mRNAs in Association with SC-35 Domains Is Gene Specific ». Journal of Cell Biology 144, n^o 4 (22 février 1999) : 617–29. http://dx.doi.org/10.1083/jcb.144.4.617.

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Analysis of six endogenous pre-mRNAs demonstrates that localization at the periphery or within splicing factor-rich (SC-35) domains is not restricted to a few unusually abundant pre-mRNAs, but is apparently a more common paradigm of many protein-coding genes. Different genes are preferentially transcribed and their RNAs processed in different compartments relative to SC-35 domains. These differences do not simply correlate with the complexity, nuclear abundance, or position within overall nuclear space. The distribution of spliceosome assembly factor SC-35 did not simply mirror the distribution of individual pre-mRNAs, but rather suggested that individual domains contain both specific pre-mRNA(s) as well as excess splicing factors. This is consistent with a multifunctional compartment, to which some gene loci and their RNAs have access and others do not. Despite similar molar abundance in muscle fiber nuclei, nascent transcript “trees” of highly complex dystrophin RNA are cotranscriptionally spliced outside of SC-35 domains, whereas posttranscriptional “tracks” of more mature myosin heavy chain transcripts overlap domains. Further analyses supported that endogenous pre-mRNAs exhibit distinct structural organization that may reflect not only the expression and complexity of the gene, but also constraints of its chromosomal context and kinetics of its RNA metabolism.

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Dale, Ryan K., Leah H. Matzat et Elissa P. Lei. « metaseq : a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA ». Nucleic Acids Research 42, n^o 14 (24 juillet 2014) : 9158–70. http://dx.doi.org/10.1093/nar/gku644.

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Abstract Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA–protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP- and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data. Based on the metaseq-enabled analysis presented here, we propose a model where Shep associates with chromatin cotranscriptionally, then is recruited to insulator complexes in trans where it plays a negative role in insulator activity.

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Lundkvist, Pär, Sara Jupiter, Åsa Segerstolpe, Yvonne N. Osheim, Ann L. Beyer et Lars Wieslander. « Mrd1p Is Required for Release of Base-Paired U3 snoRNA within the Preribosomal Complex ». Molecular and Cellular Biology 29, n^o 21 (24 août 2009) : 5763–74. http://dx.doi.org/10.1128/mcb.00428-09.

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ABSTRACT In eukaryotes, ribosomes are made from precursor rRNA (pre-rRNA) and ribosomal proteins in a maturation process that requires a large number of snoRNPs and processing factors. A fundamental problem is how the coordinated and productive folding of the pre-rRNA and assembly of successive pre-rRNA-protein complexes is achieved cotranscriptionally. The conserved protein Mrd1p, which contains five RNA binding domains (RBDs), is essential for processing events leading to small ribosomal subunit synthesis. We show that full function of Mrd1p requires all five RBDs and that the RBDs are functionally distinct and needed during different steps in processing. Mrd1p mutations trap U3 snoRNA in pre-rRNP complexes both in base-paired and non-base-paired interactions. A single essential RBD, RBD5, is involved in both types of interactions, but its conserved RNP1 motif is not needed for releasing the base-paired interactions. RBD5 is also required for the late pre-rRNP compaction preceding A2 cleavage. Our results suggest that Mrd1p modulates successive conformational rearrangements within the pre-rRNP that influence snoRNA-pre-rRNA contacts and couple U3 snoRNA-pre-rRNA remodeling and late steps in pre-rRNP compaction that are essential for cleavage at A0 to A2. Mrd1p therefore coordinates key events in biosynthesis of small ribosome subunits.

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Wong, Chi-Ming, Hongfang Qiu, Cuihua Hu, Jinsheng Dong et Alan G. Hinnebusch. « Yeast Cap Binding Complex Impedes Recruitment of Cleavage Factor IA to Weak Termination Sites ». Molecular and Cellular Biology 27, n^o 18 (16 juillet 2007) : 6520–31. http://dx.doi.org/10.1128/mcb.00733-07.

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ABSTRACT Nuclear cap binding complex (CBC) is recruited cotranscriptionally and stimulates spliceosome assembly on nascent mRNAs; however, its possible functions in regulating transcription elongation or termination were not well understood. We show that, while CBC appears to be dispensable for normal rates and processivity of elongation by RNA polymerase II (Pol II), it plays a direct role in preventing polyadenylation at weak termination sites. Similarly to Npl3p, with which it interacts, CBC suppresses the weak terminator of the gal10-Δ56 mutant allele by impeding recruitment of termination factors Pcf11p and Rna15p (subunits of cleavage factor IA [CF IA]) and does so without influencing Npl3p occupancy at the termination site. Importantly, deletion of CBC subunits or NPL3 also increases termination at a naturally occurring weak poly(A) site in the RNA14 coding sequences. We also show that CBC is most likely recruited directly to the cap of nascent transcripts rather than interacting first with transcriptional activators or the phosphorylated C-terminal domain of Pol II. Thus, our findings illuminate the mechanism of CBC recruitment and extend its function in Saccharomyces cerevisiae beyond mRNA splicing and degradation of aberrant nuclear mRNAs to include regulation of CF IA recruitment at poly(A) selection sites.

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Kiesler, Eva, Francesc Miralles et Neus Visa. « HEL/UAP56 Binds Cotranscriptionally to the Balbiani Ring Pre-mRNA in an Intron-Independent Manner and Accompanies the BR mRNP to the Nuclear Pore ». Current Biology 12, n^o 10 (mai 2002) : 859–62. http://dx.doi.org/10.1016/s0960-9822(02)00840-0.

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Raychaudhuri, G., S. R. Haynes et A. L. Beyer. « Heterogeneous nuclear ribonucleoprotein complexes and proteins in Drosophila melanogaster ». Molecular and Cellular Biology 12, n^o 2 (février 1992) : 847–55. http://dx.doi.org/10.1128/mcb.12.2.847-855.1992.

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Pre-mRNAs cotranscriptionally associate with a small group of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. We have previously identified two genes in Drosophila melanogaster, Hrb98DE and Hrb87F (i.e., genes at 98DE and 87F encoding putative hnRNA binding proteins), which encode five protein species homologous to the mammalian A-B hnRNP proteins. The studies presented herein show that antibodies against the RNP domains of Hrb98DE reacted with 10 to 15 distinct spots of 38 to 40 kDa in the basic region of two-dimensional gels. These nuclear proteins bound single-stranded nucleic acids and were extracted from Drosophila tissue culture cells as 40 to 80S hnRNP complexes in association with 300 to 800 nucleotide fragments of RNA. The peak of poly(A)+ RNA sequences was coincident with the peak of HRB proteins in sucrose gradients, strongly suggesting that the HRB complexes identified are Drosophila hnRNP complexes. The repertoire of HRB proteins did not change significantly during embryogenesis and was similar to that observed in Drosophila tissue culture cells. Analyses with peptide-specific antisera demonstrated that the major proteins in the hnRNP complex were encoded by the two genes previously identified. Although the Drosophila HRB proteins are only approximately 60% identical throughout the RNP domains to the mammalian A-B hnRNP proteins, features of the basic pre-mRNA packaging mechanism appear to be highly conserved between D. melanogaster and mammals.

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Raychaudhuri, G., S. R. Haynes et A. L. Beyer. « Heterogeneous nuclear ribonucleoprotein complexes and proteins in Drosophila melanogaster. » Molecular and Cellular Biology 12, n^o 2 (février 1992) : 847–55. http://dx.doi.org/10.1128/mcb.12.2.847.

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Pre-mRNAs cotranscriptionally associate with a small group of proteins to form heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. We have previously identified two genes in Drosophila melanogaster, Hrb98DE and Hrb87F (i.e., genes at 98DE and 87F encoding putative hnRNA binding proteins), which encode five protein species homologous to the mammalian A-B hnRNP proteins. The studies presented herein show that antibodies against the RNP domains of Hrb98DE reacted with 10 to 15 distinct spots of 38 to 40 kDa in the basic region of two-dimensional gels. These nuclear proteins bound single-stranded nucleic acids and were extracted from Drosophila tissue culture cells as 40 to 80S hnRNP complexes in association with 300 to 800 nucleotide fragments of RNA. The peak of poly(A)+ RNA sequences was coincident with the peak of HRB proteins in sucrose gradients, strongly suggesting that the HRB complexes identified are Drosophila hnRNP complexes. The repertoire of HRB proteins did not change significantly during embryogenesis and was similar to that observed in Drosophila tissue culture cells. Analyses with peptide-specific antisera demonstrated that the major proteins in the hnRNP complex were encoded by the two genes previously identified. Although the Drosophila HRB proteins are only approximately 60% identical throughout the RNP domains to the mammalian A-B hnRNP proteins, features of the basic pre-mRNA packaging mechanism appear to be highly conserved between D. melanogaster and mammals.

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Kim, Miri, Michel Bellini et Stephanie Ceman. « Fragile X Mental Retardation Protein FMRP Binds mRNAs in the Nucleus ». Molecular and Cellular Biology 29, n^o 1 (20 octobre 2008) : 214–28. http://dx.doi.org/10.1128/mcb.01377-08.

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ABSTRACT The fragile X mental retardation protein FMRP is an RNA binding protein that associates with a large collection of mRNAs. Since FMRP was previously shown to be a nucleocytoplasmic shuttling protein, we examined the hypothesis that FMRP binds its cargo mRNAs in the nucleus. The enhanced green fluorescent protein-tagged FMRP construct (EGFP-FMRP) expressed in Cos-7 cells was efficiently exported from the nucleus in the absence of its nuclear export sequence and in the presence of a strong nuclear localization sequence (the simian virus 40 [SV40] NLS), suggesting an efficient mechanism for nuclear export. We hypothesized that nuclear FMRP exits the nucleus through its bound mRNAs. Using silencing RNAs to the bulk mRNA exporter Tap/NXF1, we observed a significantly increased number of cells containing EGFP-FMRP in the nucleus, which was further augmented by removal of FMRP's nuclear export sequence. Nuclear-retained SV40-FMRP could be released upon treatment with RNase. Further, Tap/NXF1 coimmunoprecipitated with EGFP-FMRP in an RNA-dependent manner and contained the FMR1 mRNA. To determine whether FMRP binds pre-mRNAs cotranscriptionally, we expressed hemagglutinin-SV40 FMRP in amphibian oocytes and found it, as well as endogenous Xenopus FMRP, on the active transcription units of lampbrush chromosomes. Collectively, our data provide the first lines of evidence that FMRP binds mRNA in the nucleus.

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Yamasaki, Tomohito, Masayuki Onishi, Eun-Jeong Kim, Heriberto Cerutti et Takeshi Ohama. « RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii ». Proceedings of the National Academy of Sciences 113, n^o 38 (31 août 2016) : 10720–25. http://dx.doi.org/10.1073/pnas.1523230113.

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Canonical microRNAs (miRNAs) are embedded in duplexed stem–loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii. In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.

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Huang, J., et L. H. van der Ploeg. « Maturation of polycistronic pre-mRNA in Trypanosoma brucei : analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus ». Molecular and Cellular Biology 11, n^o 6 (juin 1991) : 3180–90. http://dx.doi.org/10.1128/mcb.11.6.3180-3190.1991.

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Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.

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Huang, J., et L. H. van der Ploeg. « Maturation of polycistronic pre-mRNA in Trypanosoma brucei : analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus. » Molecular and Cellular Biology 11, n^o 6 (juin 1991) : 3180–90. http://dx.doi.org/10.1128/mcb.11.6.3180.

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Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.

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