Cotranscriptional Recruitment of the U1 snRNP to Intron-Containing Genes in Yeast

Kotovic, Kimberly M.; Lockshon, Daniel; Boric, Lamia; Neugebauer, Karla M.

doi:10.1128/mcb.23.16.5768-5779.2003

Cited by 106 publications

(120 citation statements)

References 59 publications

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“…Genomic localization of RNA binding proteins identified sites that are consistent with known functions. A yeast spliceosome component, the U1 snRNP, localized preferentially to intron-containing genes, while the RNA exonuclease Rat1p localized to the 3Ј ends of genes where it is involved in transcription termination as well as mRNA degradation (Kotovic et al 2003;Kim et al 2004;Lacadie and Rosbash 2005). These studies underscore the potential for the genomic localization approach to identify functional targets of RNA binding proteins and improve our understanding of the early assembly and organization of mRNPs.…”

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confidence: 90%

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Swinburne

Meyer²,

Liu³

et al. 2006

Genome Res.

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Pre-mRNA processing often occurs in coordination with transcription thereby coupling these two key regulatory events. As such, many proteins involved in mRNA processing associate with the transcriptional machinery and are in proximity to DNA. This proximity allows for the mapping of the genomic associations of RNA binding proteins by chromatin immunoprecipitation (ChIP) as a way of determining their sites of action on the encoded mRNA. Here, we used ChIP combined with high-density microarrays to localize on the human genome three functionally distinct RNA binding proteins: the splicing factor polypyrimidine tract binding protein (PTBP1/hnRNP I), the mRNA export factor THO complex subunit 4 (ALY/THOC4), and the 3Ј end cleavage stimulation factor 64 kDa (CSTF2). We observed interactions at promoters, internal exons, and 3Ј ends of active genes. PTBP1 had biases toward promoters and often coincided with RNA polymerase II (RNA Pol II). The 3Ј processing factor, CSTF2, had biases toward 3Ј ends but was also observed at promoters. The mRNA processing and export factor, ALY, mapped to some exons but predominantly localized to introns and did not coincide with RNA Pol II. Because the RNA binding proteins did not consistently coincide with RNA Pol II, the data support a processing mechanism driven by reorganization of transcription complexes as opposed to a scanning mechanism. In sum, we present the mapping in mammalian cells of RNA binding proteins across a portion of the genome that provides insight into the transcriptional assembly of RNA-protein complexes.[Supplemental material is available online at www.genome.org and http://silver.med.harvard.edu/brodsky/.] RNA binding proteins regulate many stages of RNA metabolism to help determine gene expression programs (for reviews, see Keene and Tenenbaum 2002;Hieronymus and Silver 2004). To prepare mRNA for export to and translation in the cytoplasm, many events including splicing, cleavage of the 3Ј end, and editing occur cotranscriptionally (Kornblihtt et al. 2004;Aguilera 2005a). After transcription, the mRNA-protein complex may reorganize as it is exported to the cytoplasm and prepared for translation (Fairman et al. 2004). Thus, significant efforts have been made to determine how RNA binding proteins interact at genes and/or mRNAs at various stages of mRNA metabolism (Tenenbaum et al. 2000;Brown et al. 2001;Hieronymus and Silver 2003;Ule et al. 2003;Yu et al. 2004). These early genomic efforts suggested that, similar to DNA binding proteins, functional groupings of genes are being bound by RNA binding proteins perhaps as post-transcriptional operons (Keene and Tenenbaum 2002). However, it is not known at which stage of RNA processing these operons are established.Much of pre-mRNA processing is coupled to transcription both physically and functionally, potentially through interactions with the C-terminal domain (CTD) of RNA polymerase II (RNA Pol II) (Mortillaro et al. 1996;Yuryev et al. 1996;Kim et al. 1997). The CTD is phosphorylated as transcription begins. Following...

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confidence: 90%

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Swinburne

Meyer²,

Liu³

et al. 2006

Genome Res.

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“…In Saccharomyces cerevisiae, splicing takes place primarily during RNA Polymerase II (RNA Pol II) transcription [2][3][4] and studies have revealed that snRNPs assemble onto the nascent premRNA co-transcriptionally. [5][6][7][8][9] Mounting evidence supports a model in which pre-mRNA splicing is tightly coordinated with transcription to ensure precise and efficient gene expression. 10,11 The fact that pre-mRNA splicing occurs cotemporally with RNA Pol II-dependent gene transcription indicates that spliceosome assembly and subsequent catalytic steps take place in the context of a dynamic chromatin environment.…”

Section: Introductionmentioning

confidence: 99%

“…Previous studies indicate that all 3 of these HA-tagged proteins can associate with pre-mRNA co-transcriptionally. [5][6][7][8] We specifically monitored association of these snRNPs with the DBP2 pre-mRNA, which is the only pre-mRNA that we identified as being H3K36me3 dependent, RPS21B pre-mRNA, which is the only pre-mRNA that relies heavily on Set2 interaction with RNA Pol II for splicing, and SRB2, which requires H3 mono-and di-methylation for splicing (Fig. 6A).…”

Section: Association Ofmentioning

confidence: 99%

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

et al. 2016

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Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) -a residue methylated by Set2 during transcription elongation -exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast.Abbreviations: (H3K36), histone H3 lysine 36; (ChIP), chromatin immunoprecipitations; (RT-qPCR), reverse transcription PCR; (snRNP), small nuclear ribonucleprotein; (RNA Pol II), RNA polymerase II

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“…Previous studies demonstrate that ChIP allows detection of cotranscriptional spliceosome assembly; upon synthesis of the appropriate RNA signal, individual snRNPs appear to associate with nascent RNA in a stepwise manner (17)(18)(19)25). When spliceosomal rearrangements are perturbed, there is an observable lag in the disengagement of snRNPs with pre-mRNA, and factors that are recruited downstream of the lag show diminished association with the pre-mRNA (18,19,25) (Fig.…”

Section: Gcn5-dependent Histone H3 Acetylation Occurs Within the Codingmentioning

confidence: 99%

“…Work from several laboratories has demonstrated that the stepwise exchange of factors during cotranscriptional spliceosome assembly can be measured by ChIP (17)(18)(19). The 5′ splice site is recognized by the U1 snRNP, followed by U2 snRNP association with the branchpoint.…”

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confidence: 99%

Dynamic histone acetylation is critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements

Gunderson

Merkhofer

Johnson

2011

Proc. Natl. Acad. Sci. U.S.A.

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Assembly of the spliceosome onto pre-mRNA is a dynamic process involving the ordered exchange of snRNPs to form the catalytically active spliceosome. These ordered rearrangements have recently been shown to occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template. We previously demonstrated that the histone acetyltransferase Gcn5 is required for U2 snRNP association with the branchpoint. Here we provide evidence that histone acetylation and deacetylation facilitate proper cotranscriptional association of spliceosomal snRNPs. As with GCN5, mutation or deletion of Gcn5-targeted histone H3 residues leads to synthetic lethality when combined with deletion of the genes encoding the U2 snRNP components Lea1 or Msl1. Gcn5 associates throughout intron-containing genes and, in the absence of the histone deacetylases Hos3 and Hos2, enhanced histone H3 acetylation is observed throughout the body of genes. Deletion of histone deacetylaces also results in persistent association of the U2 snRNP and a severe defect in the association of downstream factors. These studies show that cotranscriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby yeast spliceosome assembly is tightly coupled to histone modification.pre-mRNA splicing | Saccharomyces cerevisiae R emoval of noncoding sequences from premessenger RNA is achieved by the activity of a dynamic ribonucleoprotein complex, the spliceosome. As the spliceosomal snRNPs sequentially recognize sequences in the pre-mRNA, the spliceosome undergoes ATP-dependent rearrangement of its RNA and protein components. Recently, it has been recognized that splicing can occur cotranscriptionally, while the RNA polymerase is still actively engaged with the chromatin template.The monomeric units of chromatin, nucleosomes, are comprised of DNA wrapped around the core histones H3, H4, H2A, and H2B. Histones undergo extensive posttranslational modification on their N-terminal tails that affect compaction of DNA and binding of regulatory factors. Although it is known that splicing occurs cotranscriptionally, it is far less well understood how changes in this chromatin template affect the reaction. Analysis of tiling array data has suggested that nucleosomes and, according to several of these studies, specific histone modifications are enriched in exon sequences, suggesting that there may be specific histone "marks" that are associated with splicing signals (1-7). Additionally, proteins that bind to methylated histones (H3K4me3 and H3K36me3) have been shown to facilitate the recruitment of snRNPs to the nascent transcript and influence efficiency of splicing and alternative splicing (8, 9). The chromatin-bound mammalian SWI/SNF complex associates with components of the spliceosome and affects alternative splicing (10, 11). Although these studies suggest a role for histone modifications in mammalian alternative splicing, there has been little analysis of their roles in constitu...

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Cotranscriptional Recruitment of the U1 snRNP to Intron-Containing Genes in Yeast

Cited by 106 publications

References 59 publications

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Genomic localization of RNA binding proteins reveals links between pre-mRNA processing and transcription

Histone H3K36 methylation regulates pre-mRNA splicing inSaccharomyces cerevisiae

Dynamic histone acetylation is critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements

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