Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.
Science (Impact Factor: 31.48). 01/2008; 318(5857):1777-9. DOI: 10.1126/science.1145989
Source: PubMed

ABSTRACT RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene-specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type-specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.

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Available from: Dawn O'Reilly, May 22, 2014
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    • "In this study, we demonstrated that chicken Ssu72 inactivation resulted in inefficient 3′-end formation of both snRNAs and polyadenylated mRNAs (Fig. 4), concomitant with elevation of Ser5P and Ser7P levels in the 3′ regions of these genes (Fig. 6A and 6B). Our results are in accordance with a study of cultured human cells by Egloff et al. [15] showing that substituting either the Ser5 or Ser7 residue with phospho-mimetic glutamate (Ser5E or Ser7E) in all CTD heptapeptide repeats led to defects in 3′-end formation of both snRNAs and polyadenylated mRNAs [15]. Why does elevation of either Ser5P or Ser7P levels cause the defects of 3′-end formation of these types of Pol II-transcribed genes? "
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    ABSTRACT: In eukaryotes, the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide YSPTSPS, which is subjected to reversible phosphorylation at Ser2, Ser5, and Ser7 during the transcription cycle. Dynamic changes in CTD phosphorylation patterns, established by the activities of multiple kinases and phosphatases, are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Yeast Ssu72, a CTD phosphatase specific for Ser5 and Ser7, functions in 3'-end processing of pre-mRNAs and in transcription termination of small non-coding RNAs such as snoRNAs and snRNAs. Vertebrate Ssu72 exhibits Ser5- and Ser7-specific CTD phosphatase activity in vitro, but its roles in gene expression and CTD dephosphorylation in vivo remain to be elucidated. To investigate the functions of vertebrate Ssu72 in gene expression, we established chicken DT40 B-cell lines in which Ssu72 expression was conditionally inactivated. Ssu72 depletion in DT40 cells caused defects in 3'-end formation of U2 and U4 snRNAs and GAPDH mRNA. Surprisingly, however, Ssu72 inactivation increased the efficiency of 3'-end formation of non-polyadenylated replication-dependent histone mRNA. Chromatin immunoprecipitation analyses revealed that Ssu72 depletion caused a significant increase in both Ser5 and Ser7 phosphorylation of the Pol II CTD on all genes in which 3'-end formation was affected. These results suggest that vertebrate Ssu72 plays positive roles in 3'-end formation of snRNAs and polyadenylated mRNAs, but negative roles in 3'-end formation of histone mRNAs, through dephosphorylation of both Ser5 and Ser7 of the CTD.
    PLoS ONE 08/2014; 9(8):e106040. DOI:10.1371/journal.pone.0106040 · 3.23 Impact Factor
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    • "An snRNA gene-specific complex, Integrator, is recruited to human snRNA genes to carry out 3′-end processing directed by the conserved 3′ box RNA processing element located within 19 bases downstream of the snRNA-encoding sequence (25). This complex consists of 12 subunits, 10 of which are recruited early in the transcription cycle by interaction with the pol II CTD phosphorylated on Ser7 (40,41). Later in transcription, when the pol II CTD is phosphorylated on both Ser2 and Ser7, a further two subunits, Int9 and Int11, are recruited (42). "
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    ABSTRACT: RNA polymerase II transcribes both protein coding and non-coding RNA genes and, in yeast, different mechanisms terminate transcription of the two gene types. Transcription termination of mRNA genes is intricately coupled to cleavage and polyadenylation, whereas transcription of small nucleolar (sno)/small nuclear (sn)RNA genes is terminated by the RNA-binding proteins Nrd1, Nab3 and Sen1. The existence of an Nrd1-like pathway in humans has not yet been demonstrated. Using the U1 and U2 genes as models, we show that human snRNA genes are more similar to mRNA genes than yeast snRNA genes with respect to termination. The Integrator complex substitutes for the mRNA cleavage and polyadenylation specificity factor complex to promote cleavage and couple snRNA 3'-end processing with termination. Moreover, members of the associated with Pta1 (APT) and cleavage factor I/II complexes function as transcription terminators for human snRNA genes with little, if any, role in snRNA 3'-end processing. The gene-specific factor, proximal sequence element-binding transcription factor (PTF), helps clear the U1 and U2 genes of nucleosomes, which provides an easy passage for pol II, and the negative elongation factor facilitates termination at the end of the genes where nucleosome levels increase. Thus, human snRNA genes may use chromatin structure as an additional mechanism to promote efficient transcription termination in vivo.
    Nucleic Acids Research 10/2013; 42(1). DOI:10.1093/nar/gkt892 · 9.11 Impact Factor
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    • "U1 snRNA, U2 snRNA and the RNA component of telomerase (hTR) are initially produced as longer transcripts that require 39 end processing (Jády et al., 2003; Nesic et al., 2004; Theimer et al., 2007; Zhu et al., 2004). U1 and U2 snRNA 39 end processing is facilitated by the Integrator complex in order to generate the appropriately sized mature RNA that is incorporated into the U1 or U2 snRNP (Baillat et al., 2005; Egloff et al., 2007), but it is not known how hTR processing takes place in mammalian cells. WRAP53 (also known as WDR79 or TCAB), which interacts with coilin and is required for CB formation, is required for hTR and scaRNA localization to the CB (Mahmoudi et al., 2010; Stern et al., 2012; Tycowski et al., 2009; Venteicher et al., 2009). "
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    ABSTRACT: The Cajal body (CB) is a domain of concentrated components found within the nucleus of cells in an array of species that is functionally important for the biogenesis of telomerase and small nuclear ribonucleoproteins. The CB is a dynamic structure whose number and size change during the cell cycle and is associated with other nuclear structures and gene loci. Coilin, also known as the marker protein for the CB, is a phosphoprotein widely accepted for its role in maintaining CB integrity. Recent studies have been done to further elucidate functional activities of coilin apart from its structural role in the CB in an attempt to explore the rationale for coilin expression in cells that have few CBs or lack them altogether. Here we show that the RNA association profile of coilin changes in mitosis with respect to that during interphase. We provide evidence of transcriptional and/or processing dysregulation of several CB-related RNA transcripts as a result of ectopic expression of both wild-type and phosphomutant coilin proteins. We also show apparent changes in transcription and/or processing of these transcripts upon coilin knockdown in both transformed and primary cell lines. Additionally, we provide evidence of specific coilin RNase activity regulation, on both U2 and hTR transcripts, by phosphorylation of a single residue, serine 489. Collectively, these results point to additional functions for coilin that are regulated by phosphorylation.
    Biology Open 04/2013; 2(4):407-15. DOI:10.1242/bio.20133863 · 2.42 Impact Factor
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