Article

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: 33.61). 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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    • "The CTD interacts with a wide range of nuclear factors based on its dynamic plasticity and diversity of binding surfaces (Jasnovidova and Stefl, 2013;Meinhart et al., 2005;Phatnani and Greenleaf, 2006) generated by differential phosphorylation at multiple sites, a phenomenon that was referred to as the ''CTD code'' (Buratowski, , 2009). Monoclonal antibodies have been used to study in vivo the potentially phosphorylated CTD amino acids (Y 1 , S 2 , T 4 , S 5 , and S 7 ) (Chapman et al., 2007;Descostes et al., 2014;Egloff et al., 2007;Hintermair et al., 2012;Hsin et al., 2011;Lu et al., 1992;Mayer et al., 2012;Zhang and Corden, 1991). However, the epitopes detected by antibodies can be masked by proteins or modifications at neighboring sites. "
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    ABSTRACT: The carboxy-terminal domain (CTD) of RNA polymerase II (Pol II) consists of heptad repeats with the consensus motif Y1-S2-P3-T4-S5-P6-S7. Dynamic phosphorylation of the CTD coordinates Pol II progression through the transcription cycle. Here, we use genetic and mass spectrometric approaches to directly detect and map phosphosites along the entire CTD. We confirm phosphorylation of CTD residues Y1, S2, T4, S5, and S7 in mammalian and yeast cells. Although specific phosphorylation signatures dominate, adjacent CTD repeats can be differently phosphorylated, leading to a high variation of coexisting phosphosites in mono- and di-heptad CTD repeats. Inhibition of CDK9 kinase specifically reduces S2 phosphorylation levels within the CTD.
    Full-text · Article · Jan 2016 · Molecular Cell
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    • "Fusing two noncoding RNAs into a chimeric primary transcript may be a strategy used by the virus because of its limited genome size. It also seems reasonable for the virus to hijack the host Integrator complex, which is recruited to all snRNA gene promoters (Egloff et al. 2007), to execute a second cleavage of the same substrate, thereby producing both the 5 ′ and 3 ′ ends of the HVS pre-miRNAs. Notably, Integrator-generated pre-miR-HUSR4 has noncanonical 5 ′ and 3 ′ overhangs compared with Microprocessor-generated pre-miRNAs, which contain 2-nt 3 ′ overhangs (Fig. 1B; Cazalla et al. 2011). "
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    ABSTRACT: Herpesvirus saimiri (HVS) is an oncogenic γ-herpesvirus that produces microRNAs (miRNAs) by cotranscription of precursor miRNA (pre-miRNA) hairpins immediately downstream from viral small nuclear RNAs (snRNA). The host cell Integrator complex, which recognizes the snRNA 3' end processing signal (3' box), generates the 5' ends of HVS pre-miRNA hairpins. Here, we identify a novel 3' box-like sequence (miRNA 3' box) downstream from HVS pre-miRNAs that is essential for miRNA biogenesis. In vivo knockdown and rescue experiments confirmed that the 3' end processing of HVS pre-miRNAs also depends on Integrator activity. Interaction between Integrator and HVS primary miRNA (pri-miRNA) substrates that contain only the miRNA 3' box was confirmed by coimmunoprecipitation and an in situ proximity ligation assay (PLA) that we developed to localize specific transient RNA-protein interactions inside cells. Surprisingly, in contrast to snRNA 3' end processing, HVS pre-miRNA 3' end processing by Integrator can be uncoupled from transcription, enabling new approaches to study Integrator enzymology. © 2015 Xie et al.; Published by Cold Spring Harbor Laboratory Press.
    Full-text · Article · Jul 2015 · Genes & development
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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.
    Full-text · Article · Aug 2014 · PLoS ONE
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