Tat Stimulates Cotranscriptional Capping of HIV mRNA

Chiu, Ya‐Lin; Ho, Caleb; Saha, Nayanendu; Schwer, Beate; Shuman, Stewart; Rana, Tariq M.

doi:10.1016/s1097-2765(02)00630-5

Cited by 128 publications

(126 citation statements)

References 27 publications

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“…We surmise that the active kinase is a heterodimer of the 68-kDa SpCdk9 and 43-kDa Pch1 polypeptides. DISCUSSION mRNA capping is coupled to transcription elongation via physical interactions between the cap-forming enzymes, the phosphorylated pol II CTD, and the elongation factor Spt5 (5,6,17,27). Here we report that fission yeast RNA triphosphatase, the enzyme that initiates cap formation, interacts with SpCdk9, an ortholog of the Cdk9 subunit of metazoan P-TEFb.…”

Section: Figmentioning

confidence: 87%

“…In the case of HIV, Spt5-induced arrest at promoter-proximal sites would maximize the oppor-tunity for recruitment of Mce1 to the elongation complex, by a multiplicity of Mce1 interactions with Tat, Spt5, and/or pol II CTD-PO 4 . Recent studies show that Tat directly stimulates the cotranscriptional capping of nascent HIV pre-mRNA (17). An inference is that Tat serves a dual role in promoting capping during the transcription arrest and in activating P-TEFb to then override the elongation block.…”

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confidence: 99%

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Interactions between Fission Yeast Cdk9, Its Cyclin Partner Pch1, and mRNA Capping Enzyme Pct1 Suggest an Elongation Checkpoint for mRNA Quality Control

Pei¹,

Schwer²,

Shuman³

2003

Journal of Biological Chemistry

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RNA polymerase II (pol II) is subject to an early elongation delay induced by negative factors Spt5/Spt4 and NELF, which is overcome by the positive factor P-TEFb (Cdk9/cyclin T), a protein kinase that phosphorylates the pol II C-terminal domain (CTD) and the transcription elongation factor Spt5. Although the rationale for this arrest and restart is unclear, recent studies suggest a connection to mRNA capping, which is coupled to transcription elongation via physical and functional interactions between the cap-forming enzymes, the CTD-PO 4 , and Spt5. Here we identify a novel interaction between fission yeast RNA triphosphatase Pct1, the enzyme that initiates cap formation, and Schizosaccharomyces pombe Cdk9. The C-terminal segment of SpCdk9 comprises a Pct1-binding domain distinct from the Nterminal Cdk domain. We show that the Cdk domain interacts with S. pombe Pch1, a homolog of cyclin T, and that the purified recombinant SpCdk9/Pch1 heterodimer can phosphorylate both the pol II CTD and the C-terminal domain of S. pombe Spt5. We provide genetic evidence that SpCdk9 and Pch1 are functional orthologs of the Saccharomyces cerevisiae CTD kinase Bur1/Bur2, a putative yeast P-TEFb. Mutations of the kinase active site and the regulatory T-loop of SpCdk9 abolish its activity in vivo. Deleting the C-terminal domain of SpCdk9 causes a severe growth defect. We suggest a model whereby Spt5-induced arrest of early elongation ensures a temporal window for recruitment of the capping enzymes, which in turn attract Cdk9 to alleviate the arrest. This elongation checkpoint may avoid wasteful rounds of transcription of uncapped pre-mRNAs.The 5Ј-cap is the defining structural feature of eukaryotic mRNA. Consisting of m 7 G linked via an inverted 5Ј-5Ј triphosphate bridge to the initiating nucleoside of the transcript, the cap is formed by enzymatic modification of pre-mRNAs as they are being synthesized by RNA polymerase II (pol II). 1 Capping entails three reactions: (i) the 5Ј-triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase, (ii) the diphosphate RNA end is capped with GMP by RNA guanylyltransferase, and (iii) the GpppN cap is methylated by RNA (guanine-N7) methyltransferase (1). Targeting of cap formation to pre-mRNAs depends on interactions of the capping enzymes with the phosphorylated C-terminal domain (CTD) of the largest subunit of pol II (Ref. 2 and citations therein). Recruitment of the capping apparatus to the elongation complex requires the TFIIH-associated CTD kinase (Kin28 in yeast, Cdk7 in mammals), which phosphorylates Ser-5 of the CTD heptad repeat YSPTSPS (3, 4).Other protein-protein contacts may also be involved in coupling capping to pol II transcription elongation. The pol II elongation factor Spt5 binds directly to the triphosphatase and guanylyltransferase components of the mammalian and Schizosaccharomyces pombe capping apparatus (5, 6). The fission yeast S. pombe employs a distinctive strategy of cap targeting whereby the triphosphatase (Pct1) and guanylyltrans...

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Section: Figmentioning

confidence: 87%

mentioning

confidence: 99%

Interactions between Fission Yeast Cdk9, Its Cyclin Partner Pch1, and mRNA Capping Enzyme Pct1 Suggest an Elongation Checkpoint for mRNA Quality Control

Pei¹,

Schwer²,

Shuman³

2003

Journal of Biological Chemistry

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“…Capped pol II transcripts as short as 25-40 bases are made in transcription extracts and isolated nuclei suggesting that capping is a very early co-transcriptional event 14 -17. Both HCE and MT can bind directly to pol II that is phosphorylated on the CTD 18 but in vitro studies suggest that these interactions with actively transcribing pol II are weak 16,17 , and their in vivo significance remains unknown.…”

Section: Introductionmentioning

confidence: 99%

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

Glover-Cutter

Kim

Espinosa

et al. 2007

Nat Struct Mol Biol

302

336

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We investigated co-transcriptional recruitment of pre-mRNA processing factors to human genes. Capping factors associate with paused RNA pol II at the 5′ ends of quiescent genes. They also track throughout actively transcribed genes, and accumulate with paused polymerase in the 3′ flanking region. 3′ processing factors CstF and CPSF are maximally recruited 0.5-1.5 kb downstream of poly (A) sites where they coincide with capping factors, Spt5, and Ser2 hyperphosphorylated, paused pol II. 3′ end processing factors also localize at transcription start sites, and this early recruitment is enhanced after polymerase arrest with DRB. These results suggest that promoters may help specify recruitment of 3′ end processing factors. We propose a dual pausing model where elongation arrests near the transcription start site and in the 3′ flank to allow co-transcriptional processing by factors recruited to the pol II ternary complex.

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“…These and other observations underlie the proposal of a transcription elongation checkpoint that ensures a temporal window for capping of nascent mRNAs Mandal et al 2004;Sims et al 2004). Evidence is emerging that positive and negative regulation of transcription can occur at the step of capping enzyme recruitment (Chiu et al 2002;Gao and Gross 2008).…”

Section: Introductionmentioning

confidence: 69%

“…The earliest processing step is mRNA capping, which can occur as soon as the 59 triphosphate terminus of the nascent RNA extrudes from the elongating RNA polymerase (Chiu et al 2002). The capping enzymes are directed to nascent mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II McCracken et al 1997;Yue et al 1997;Ho and Shuman 1999;Pei et al 2001;Fabrega et al 2003).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

Schwer

Schneider²,

Pei³

et al. 2009

RNA

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The Spt5-Spt4 complex regulates early transcription elongation by RNA polymerase II and has an imputed role in pre-mRNA processing via its physical association with mRNA capping enzymes. Here we characterize the Schizosaccharomyces pombe core Spt5-Spt4 complex as a heterodimer and map a trypsin-resistant Spt4-binding domain within the Spt5 subunit. A genetic analysis of Spt4 in S. pombe revealed it to be inessential for growth at 25°C-30°C but critical at 37°C. These results echo the conditional spt4D growth phenotype in budding yeast, where we find that Saccharomyces cerevisiae and S. pombe Spt4 are functionally interchangeable. Complementation of S. cerevisiae spt4D and a two-hybrid assay for Spt4-Spt5 interaction provided a readout of the effects of 33 missense and truncation mutations on S. pombe Spt4 function in vivo, which were interpreted in light of the recent crystal structure of S. cerevisiae Spt4 fused to a fragment of Spt5. Our results highlight the importance of the Spt4 Zn 2+ -binding residues-Cys12, Cys15, Cys29, and Asp32-and of Ser57, a conserved constituent of the Spt4-Spt5 interface. The 990-amino acid S. pombe Spt5 protein has an exceptionally regular carboxyl-terminal domain (CTD) composed of 18 nonapeptide repeats. We find that as few as three nonamer repeats sufficed for S. pombe growth, but only when Spt4 was present. Synthetic lethality of the spt5 1-835 spt4D double mutant at 34°C suggests that interaction of Spt4 with the central domain of Spt5 overlaps functionally with the Spt5 CTD.

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Tat Stimulates Cotranscriptional Capping of HIV mRNA

Cited by 128 publications

References 27 publications

Interactions between Fission Yeast Cdk9, Its Cyclin Partner Pch1, and mRNA Capping Enzyme Pct1 Suggest an Elongation Checkpoint for mRNA Quality Control

Interactions between Fission Yeast Cdk9, Its Cyclin Partner Pch1, and mRNA Capping Enzyme Pct1 Suggest an Elongation Checkpoint for mRNA Quality Control

RNA polymerase II pauses and associates with pre-mRNA processing factors at both ends of genes

Characterization of the Schizosaccharomyces pombe Spt5-Spt4 complex

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