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

Lindstrom, Derek L.; Squazzo, Sharon L.; Muster, Nemone; Burckin, Todd; Wächter, Kristin; Emigh, C. A.; Mccleery, J. A.; Yates, John R.; Hartzog, Grant A.

doi:10.1128/mcb.23.4.1368-1378.2003

Cited by 251 publications

(289 citation statements)

References 67 publications

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“…Thus the MED31/SOH1 subunit of the Mediator displays genetic interactions not only with Pol II (34), but also with TFIIB (34) and several transcription elongation factors that include SII (35), the last in turn displaying close relationships with hSpt4-hSpt5/DSIF (36)(37)(38)(39). Furthermore, consistent with its role in early elongation, TFIIB has also been connected to diverse elongation and processing factors (40).…”

Section: Discussionmentioning

confidence: 90%

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Malik

Barrero

Jones

2007

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

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The multiprotein Mediator coactivator complex is universally required for transcription of metazoan genes. It has been proposed to function by interfacing between transcriptional activators and the RNA polymerase II machinery. However, in vitro transcription systems reconstituted from homogeneous preparations of RNA polymerase II, the general transcription initiation factors, and the cofactor PC4 display relatively robust activator (HNF-4)-dependent activity, which, nonetheless, can be further stimulated by Mediator. By contrast, an unfractionated nuclear extract-based system in which Mediator has been immunodepleted displays a nearabsolute dependence on ectopic Mediator. Here, we identified and purified an activity, MSA-2, that confers extract-like Mediator responsiveness to our reconstituted system. Mass spectrometric analyses identified its two constituent polypeptides as hSpt5 and hSpt4, which also comprise the elongation factor DSIF. Mechanistically, MSA-2/DSIF acts by restricting overall transcription in the pure system, thereby imposing a strong Mediator dependence. Our data thus point to potential mechanisms for Mediator function beyond its presently believed role in promoting the initial formation of the RNA polymerase II-containing preinitiation complex.DRB sensitivity-inducing factor ͉ hepatocyte nuclear factor-4 ͉ positive cofactor 2 ͉ RNA polymerase II

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Section: Discussionmentioning

confidence: 90%

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Malik

Barrero

Jones

2007

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

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“…Following initiation, RNA Pol II transcribes at varying rates and undergoes conformational changes during transcript elongation that may result in pausing at particular loci (for review, see Arndt and Kane 2003). Many connections between transcription and RNA processing have been found to occur during transcription elongation (Howe et al 2003;Lindstrom et al 2003). A recent study suggested cotranscriptional specificity for RNA binding proteins; some splicing factors were constitutively recruited while others depended on the splicing outcome (Mabon and Misteli 2005).…”

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confidence: 99%

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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“…Spt5 binds directly to the triphosphatase and guanylyltransferase components of the capping apparatus in mammals and in the fission yeast S. pombe (27,28). Connections between capping enzymes and Spt5 have also been reported in S. cerevisiae (29). The interactions of the S. pombe triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes have been studied in vivo and in vitro using twohybrid assays and purified recombinant proteins, respectively (28).…”

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confidence: 99%

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

Pei

Shuman

2003

Journal of Biological Chemistry

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Schizosaccharomyces pombe Cdk9/Pch1 protein kinase is a functional ortholog of the essential Saccharomyces cerevisiae Bur1/Bur2 kinase and a putative ortholog of metazoan P-TEFb (Cdk9/cyclin T). SpCdk9/Pch1 phosphorylates of the carboxyl-terminal domain (CTD) of the S. pombe transcription elongation factor Spt5, which consists of 18 tandem repeats of a nonapeptide of consensus sequence 1 TPAWNSGSK 9 . We document the divalent cation dependence and specificity of SpCdk9/Pch1, its NTP dependence and specificity, the dependence of Spt5-CTD phosphorylation on the number of tandem nonamer repeats, and the specificity for phosphorylation of the Spt5-CTD on threonine at position 1 within the nonamer element. SpCdk9/Pch1 also phosphorylates the CTD heptaptide repeat array of the largest subunit of S. pombe RNA polymerase II (consensus sequence YSPTSPS) and does so exclusively on serine. SpCdk9/Pch1 catalyzes autophosphorylation of the kinase and cyclin subunits of the kinase complex. The distribution of phosphorylation sites on SpCdk9 (86% Ser(P), 11% Thr(P), 3% Tyr(P)) is distinct from that on Pch1 (2% Ser(P), 98% Thr(P)). We conducted a structure-guided mutational analysis of SpCdk9, whereby a total of 29 new mutations of 12 conserved residues were tested for in vivo function by complementation of a yeast bur1⌬ mutant. We identified many lethal and conditional mutations of side chains implicated in binding ATP and the divalent cation cofactor, phosphoacceptor substrate recognition, and T-loop dynamics. We surmise that the lethality of the of T212A mutation in the T-loop reflects an essential phosphorylation event, insofar as the conservative T212S change rescued wild-type growth; the phosphomimetic T212E change rescued growth at 30°C; and the effects of mutating the T-loop threonine were phenocopied by mutations in the three conserved arginines predicted to chelate the phosphate on the T-loop threonine.mRNA synthesis by RNA polymerase (pol) 1 II is regulated by phosphorylation of several of the protein components of the transcription apparatus (1). Phosphorylation of the carboxylterminal domain (CTD) of the largest subunit of pol II has been the focus of much attention because the CTD acts as an essential scaffold for the binding of macromolecular assemblies that regulate mRNA synthesis and cotranscriptional mRNA processing (2). The pol II CTD is composed of a heptapeptide motif of consensus sequence YSPTSPS repeated in tandem. The mammalian pol II CTD has 52 heptad repeats, the fission yeast Schizosaccharomyces pombe CTD has 29 repeats, and the budding yeast Saccharomyces cerevisiae CTD has 26 copies. The pol II CTD undergoes a cycle of extensive phosphorylation and dephosphorylation at positions Ser 5 and Ser 2 , which is coordinated with the transcription cycle (3). Multiple CTD kinases are present in eukaryotic cells, e.g. budding yeast has four CTD kinases, two of which (Kin28 and Bur1) are essential (3). CTD kinases are heteromeric enzymes consisting of a cyclin subunit plus a cyclin-dependent kinase (Cdk) subun...

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

Cited by 251 publications

References 67 publications

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

Identification of a regulator of transcription elongation as an accessory factor for the human Mediator coactivator

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

Characterization of the Schizosaccharomyces pombe Cdk9/Pch1 Protein Kinase

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