Functional Coupling of Cleavage and Polyadenylation with Transcription of mRNA

Abstract

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...

Concurrent splicing and transcription are not sufficient to enhance splicing efficiency

“…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).…”

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

“…There are several precedents for viral and cellular capping enzymes influencing transcription initiation, elongation and termination 24 , 40 ,41 . It is also of interest that HCE promoted poly (A) site cleavage in a transcriptioncoupled in vitro system 42 . The recruitment patterns we observed for human capping enzymes differ from their yeast counterparts.…”

Formation of the 3′ end of histone mRNA: Getting closer to the end

“…The coupling between transcription and cleavage/polyadenylation is believed to improve the efficiency and accuracy of 3' end processing by increasing the local concentration of processing factors in the vicinity of the nascent transcript. Indeed, a significantly higher rate of cleavage coupled to transcription compared to cleavage of a pre-synthesized RNA substrate was recently recapitulated in vitro (Adamson et al, 2005). The CTD is also required for the cleavage reaction in an uncoupled in vitro system, suggesting that it may play a more general function in 3' end processing (Hirose and Manley, 1998).…”