Functional Coupling of Capping and Transcription of mRNA

Moteki, Shin A.; Price, David H.

doi:10.1016/s1097-2765(02)00660-3

Cited by 124 publications

(140 citation statements)

References 42 publications

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“…In the past few years successful attempts were made to develop systems that coupled transcription and capping (Moteki and Price 2002), as well as transcription and polyadenylation (Adamson et al 2005;Rigo et al 2005). In vitro systems that combine transcription and splicing have also been described (Ghosh and GarciaBlanco 2000;Ibrahim et al 2005;Das et al 2006).…”

Section: Resultsmentioning

confidence: 99%

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Lazarev

Manley²

2007

RNA

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The concept of a tight integration of transcription and splicing of mRNA precursors has been supported with increasing evidence in recent years. However, the mechanism and functional consequences of this integration remain largely unknown. We have examined how these processes impact upon one another when they occur together in HeLa nuclear extract. While both processes do in fact occur in parallel reactions in the extracts, we found no evidence that one process affects the other, under a variety of conditions tested. For example, neither the kinetics nor efficiency of splicing is significantly enhanced by de novo RNA polymerase II-mediated transcription, relative to that of presynthesized RNA added exogenously to the extract. Our results indicate that the act of transcription by RNA polymerase II in vitro is not sufficient to enhance splicing of the newly made RNA.

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Section: Resultsmentioning

confidence: 99%

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Lazarev

Manley²

2007

RNA

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“…The cleavage site was 180 bp downstream of the start site of transcription and was followed by about 900 bp of template that could be used to stall polymerases after they had passed the site of processing. The cytomegalovirus promoter was chosen because it has been extensively used to study the function of the elongation control factors, P-TEFb, NELF, and DSIF (29,30), a termination factor, TTF2 (31), and the functional coupling of the capping enzyme, HCE, with transcription (27).…”

Section: Resultsmentioning

confidence: 99%

“…One such human system was used to examine capping during transcription and it was found that the transcription complex had a profound influence on the capping reaction (27). We have adapted that transcription system to the study of polyadenylation and have begun to define parameters of the coupling process.…”

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confidence: 99%

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Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Adamson

Shutt

Price

2005

Journal of Biological Chemistry

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Cleavage and polyadenylation define the 3 ends of almost all eukaryotic mRNAs and are thought to occur during transcription. We describe a human in vitro system utilizing an immobilized template, in which transcripts in RNA polymerase II elongation complexes are efficiently cleaved and polyadenylated. Because the cleavage rate of free RNA is much slower, we conclude that cleavage is functionally coupled to transcription. Inhibition of positive transcription elongation factor b (P-TEFb) had only a modest negative effect on cleavage, as long as transcripts were long enough to contain the polyadenylation signal. In contrast, removal of the carboxyl-terminal domain of the large subunit of RNA polymerase II had a dramatic negative effect on cleavage. Unexpectedly, the 5 portion of transcript after cleavage remained associated with the template in a functional, polyadenylation-competent complex. Efficient cleavage required 5 capping by the human capping enzyme, but the reduction of cleavage seen of transcripts in COOH-terminal domain-less polymerase elongation complexes, was not because of lack of capping.Processing of eukaryotic mRNA starts during transcription and is influenced by the RNA polymerase II elongation complex (1, 2). Capping, polyadenylation, and splicing have been seen to occur on nascent transcripts in vitro, and a variety of in vivo and in vitro approaches have strongly implicated the carboxyl-terminal domain (CTD) 2 of the large subunit of RNA polymerase II in connecting transcription with these events (3-6). Whereas many of the factors required for mRNA processing have been identified and characterized, much less is known about how the processing and transcription machinery functionally influence each other.3Ј End formation is linked to transcription. Although RNA polymerase II is capable of transcribing hundreds of kilobase pairs in a completely processive manner, after transcribing a functional polyadenylation signal the polymerase usually terminates within 1 kb (3, 7) in a process that requires the CTD (8, 9). Factors required for polyadenylation have been found to associate with isolated transcription complexes (10) and the CTD has been implicated in bringing in the factors (11, 12). There is some controversy about when polyadenylation factors associate with the transcription complex. Polyadenylation factors have been found to associate with promoter binding factors (13), and yeast chromatin immunoprecipitation experiments in one study have localized the factors throughout the gene (11), but in another only at the 3Ј end of genes after the passage of a functional polyadenylation signal (14). Recent studies in yeast (15), Drosophila (16), and Xenopus (17) have implicated the CTD kinase positive transcription elongation factor b (P-TEFb) in promoting efficient polyadenylation. Drosophila histone mRNA 3Ј end formation, involving a completely different set of factors, was found not to be stimulated by having the RNA in an elongation complex (18). However, a strong transcriptional pause was found a...

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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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Functional Coupling of Capping and Transcription of mRNA

Cited by 124 publications

References 42 publications

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

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

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