Terminating the transcript: breaking up is hard to do.
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Allosteric Regulation↗
Biomedical subjects
Emanuel Rosonina
Publications and source records attributed to Emanuel Rosonina.
From transcription to mRNA: PAF provides a new path.
Two new studies reveal a novel connection between transcription and pre-mRNA processing: the Paf1 elongation complex is involved in the 3' end formation of polyadenylated and nonpolyadenylated RNA polymerase II transcripts.
Gene Expression Regulation↗
Role for PSF in mediating transcriptional activator-dependent stimulation of pre-mRNA processing in vivo.
In a recent study, we provided evidence that strong promoter-bound transcriptional activators result in higher levels of splicing and 3'-end cleavage of nascent pre-mRNA than do weak promoter-bound activators and that this effect of strong activators requires the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II). In the present study, we have investigated the mechanism of activator- and CTD-mediated stimulation of pre-mRNA processing. Affinity chromatography experiments reveal that two factors previously implicated in the coupling of transcription and pre-mRNA processing, PSF and p54(nrb)/NonO, preferentially bind a strong rather than a weak activation domain. Elevated expression in human 293 cells of PSF bypasses the requirement for a strong activator to promote efficient splicing and 3'-end cleavage. Truncation of the pol II CTD, which consists of 52 repeats of the consensus heptapeptide sequence YSPTSPS, to 15 heptapeptide repeats prevents PSF-dependent stimulation of splicing and 3'-end cleavage. Moreover, PSF and p54(nrb)/NonO bind in vitro to the wild-type CTD but not to the truncated 15-repeat CTD, and domains in PSF that are required for binding to activators and to the CTD are also important for the stimulation of pre-mRNA processing. Interestingly, activator- and CTD-dependent stimulation of splicing mediated by PSF appears to primarily affect the removal of first introns. Collectively, these results suggest that the recruitment of PSF to activated promoters and the pol II CTD provides a mechanism by which transcription and pre-mRNA processing are coordinated within the cell.
Cell Line↗
Analysis of the requirement for RNA polymerase II CTD heptapeptide repeats in pre-mRNA splicing and 3'-end cleavage.
The carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) plays an important role in coupling transcription with precursor messenger RNA (pre-mRNA) processing. Efficient capping, splicing, and 3'-end cleavage of pre-mRNA depend on the CTD. Moreover, specific processing factors are known to associate with this structure. The CTD is therefore thought to act as a platform that facilitates the assembly of complexes required for the processing of nascent transcripts. The mammalian CTD contains 52 tandemly repeated heptapeptides with the consensus sequence YSPTSPS. The C-terminal half of the mammalian CTD contains mostly repeats that diverge from this consensus sequence, whereas the N-terminal half contains mostly repeats that match the consensus sequence. Here, we demonstrate that 22 tandem repeats, from either the conserved or divergent halves of the CTD, are sufficient for approximate wild-type levels of transcription, splicing, and 3'-end cleavage of two different pre-mRNAs, one containing a constitutively spliced intron, and the other containing an intron that depends on an exon enhancer for efficient splicing. In contrast, each block of 22 repeats is not sufficient for efficient inclusion of an alternatively spliced exon in another pre-mRNA. In this case, a longer CTD is important for counteracting the negative effect of a splicing silencer element located within the alternative exon. Our results indicate that the length, rather than the composition of CTD repeats, can be the major determinant in efficient processing of different pre-mRNA substrates. However, the extent of this length requirement depends on specific sequence features within the pre-mRNA substrate.
Alternative Splicing↗
Transcriptional activators control splicing and 3'-end cleavage levels.
We have investigated whether transcriptional activators influence the efficiency of constitutive splicing and 3'-end formation, in addition to transcription levels. Remarkably, strong activators result in higher levels of splicing and 3'-cleavage than weak activators and can control the efficiency of these steps in pre-mRNA processing separately. The pre-mRNA processing stimulatory property of activators is dependent on their binding to promoters, but is not an indirect consequence of the levels of transcripts produced. Moreover, stimulation of splicing and cleavage by a strong activator operates by a mechanism that requires the carboxyl-terminal domain of RNA polymerase II. The splicing stimulatory property of activators was observed for unrelated transcripts and for separate introns within a transcript, indicating a possible general role for strong activators in facilitating pre-mRNA processing levels. The results suggest that the efficiency of constitutive splicing and 3'-end cleavage is closely coordinated with transcription levels by promoter-bound activators.
Alternative Splicing↗
Gene expression: the close coupling of transcription and splicing.
Increasing evidence indicates that the transcriptional machinery can influence the efficiency of splicing as well as splice-site selection. Surprisingly, it has now been demonstrated that splicing components influence the efficiency of transcription. This mutual stimulation has important implications for the regulation of gene expression.
Gene Expression Regulation↗