Dual Roles for Spt5 in Pre-mRNA Processing and Transcription Elongation Revealed by Identification of Spt5-Associated Proteins

Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, California 95064, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 03/2003; 23(4):1368-78. DOI: 10.1128/MCB.23.4.1368-1378.2003
Source: PubMed


During transcription elongation, eukaryotic RNA polymerase II (Pol II) must contend with the barrier presented by nucleosomes.
The conserved Spt4-Spt5 complex has been proposed to regulate elongation through nucleosomes by Pol II. To help define the
mechanism of Spt5 function, we have characterized proteins that coimmunopurify with Spt5. Among these are the general elongation
factors TFIIF and TFIIS as well as Spt6 and FACT, factors thought to regulate elongation through nucleosomes. Spt5 also coimmunopurified
with the mRNA capping enzyme and cap methyltransferase, and spt4 and spt5 mutations displayed genetic interactions with mutations in capping enzyme genes. Additionally, we found that spt4 and spt5 mutations lead to accumulation of unspliced pre-mRNA. Spt5 also copurified with several previously unstudied proteins; we
demonstrate that one of these is encoded by a new member of the SPT gene family. Finally, by immunoprecipitating these factors we found evidence that Spt5 participates in at least three Pol
II complexes. These observations provide new evidence of roles for Spt4-Spt5 in pre-mRNA processing and transcription elongation.

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    • "Both Puf6 and Loc1 are thought to be recruited post-transcriptionally during a nucleolar transit of the ASH1 mRNA mediated by She2 (12). However, both Puf6 and Loc1 are also associated with the transcription elongation factor Spt5 in vivo (14), suggesting that Puf6 and/or Loc1 may instead be loaded on mRNA during transcription. "
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    ABSTRACT: Messenger RNA (mRNA) localization is coupled to the translational repression of transcripts during their transport. It is still unknown if this coupling depends on physical interactions between translational control and mRNA localization machineries, and how these interactions are established at the molecular level. In yeast, localization of transcripts like ASH1 to the bud depends on the RNA-binding protein She2. During its transport, ASH1 mRNA translation is repressed by Puf6. Herein, we report that She2 recruits Puf6 on ASH1 co-transcriptionally. The recruitment of Puf6 depends on prior co-transcriptional loading of Loc1, an exclusively nuclear protein. These proteins form a ternary complex, in which Loc1 bridges Puf6 to She2, that binds the ASH1 3′UTR. Using a genome-wide ChIP-chip approach, we identified over 40 novel targets of Puf6, including several bud-localized mRNAs. Interestingly, the co-transcriptional recruitment of Puf6 on genes coding for these bud-localized mRNAs is also She2- and Loc1-dependent. Our results suggest a coordinated assembly of localization and translational control machineries on localized mRNAs during transcription, and underline the importance of co-transcriptional events in establishing the cytoplasmic fate of mRNAs.
    Nucleic Acids Research 07/2014; 42(13). DOI:10.1093/nar/gku597 · 9.11 Impact Factor
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    • "This result was consistent with a prior study that showed bur2 deletions decrease H3K36 methylation (48), and with our finding that Set2 was also reduced in this strain (Figure 4G). As BUR also phosphorylates the C-terminal repeat domain of Spt5, we asked if this domain might be involved in the regulation of the Spt6–Ctk1 loop, perhaps through the recruitment of PAF complex (64). Deletion of the C-terminal repeat domain in Spt5 did not have any appreciable effect on the levels of Ser2 CTD phosphorylation, Spt6, Set2, H3K36 methylation or any other histone modification tested (Supplementary Figure S3). "
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    ABSTRACT: The C-terminal domain (CTD) of RNA polymerase II is sequentially modified for recruitment of numerous accessory factors during transcription. One such factor is Spt6, which couples transcription elongation with histone chaperone activity and the regulation of H3 lysine 36 methylation. Here, we show that CTD association of Spt6 is required for Ser2 CTD phosphorylation and for the protein stability of Ctk1 (the major Ser2 CTD kinase). We also find that Spt6 associates with Ctk1, and, unexpectedly, Ctk1 and Ser2 CTD phosphorylation are required for the stability of Spt6-thus revealing a Spt6-Ctk1 feed-forward loop that robustly maintains Ser2 phosphorylation during transcription. In addition, we find that the BUR kinase and the polymerase associated factor transcription complex function upstream of the Spt6-Ctk1 loop, most likely by recruiting Spt6 to the CTD at the onset of transcription. Consistent with requirement of Spt6 in histone gene expression and nucleosome deposition, mutation or deletion of members of the Spt6-Ctk1 loop leads to global loss of histone H3 and sensitivity to hydroxyurea. In sum, these results elucidate a new control mechanism for the regulation of RNAPII CTD phosphorylation during transcription elongation that is likely to be highly conserved.
    Nucleic Acids Research 10/2013; 42(2). DOI:10.1093/nar/gkt1003 · 9.11 Impact Factor
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    • "Downregulation of DSIF Differentially Affects mRNA Capping of NF-kB Target Genes Spt5 was reported to interact with capping enzymes (Lindstrom et al., 2003; Mandal et al., 2004; Pei and Shuman, 2002; Wen and Shatkin, 1999), so we first examined the role of DSIF on cotranscriptional capping of A20, IkBa, JunB, and CXCL1. We used an antibody that specifically recognizes the m7G cap structure (Moteki and Price, 2002) to immunoprecipitate capped mRNAs from TNF-a-stimulated control or Spt5 knockdown cells (Fig- ure "
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    ABSTRACT: NF-κB is central for immune response and cell survival, and its deregulation is linked to chronic inflammation and cancer through poorly defined mechanisms. IκBα and A20 are NF-κB target genes and negative feedback regulators. Upon their activation by NF-κB, DSIF is recruited, P-TEFb is released, and their elongating polymerase II (Pol II) C-terminal domain (CTD) remains hypophosphorylated. We show that upon DSIF knockdown, mRNA levels of a subset of NF-κB targets are not diminished; yet much less IκBα and A20 protein are synthesized, and NF-κB activation is abnormally prolonged. Further analysis of IκBα and A20 mRNA revealed that a significant portion is uncapped, unspliced, and retained in the nucleus. Interestingly, the Spt5 C-terminal repeat (CTR) domain involved in elongation stimulation through P-TEFb is dispensable for IκBα and A20 regulation. These findings assign a function for DSIF in cotranscriptional mRNA processing when elongating Pol II is hypophosphorylated and define DSIF as part of the negative feedback regulation of NF-κB.
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