Emanuel Rosonina

Role for PSF in Mediating Transcriptional Activator-Dependent Stimulation of Pre-mRNA Processing In Vivo

ABSTRACT 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 p54nrb/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 p54nrb/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.

SUMO functions in constitutive transcription and during activation of inducible genes in yeast

Transcription factors represent one of the largest groups of proteins regulated by SUMO (small ubiquitin-like modifier) modification, and their sumoylation is usually associated with transcriptional repression. To investigate whether sumoylation plays a general role in regulating transcription in yeast, we determined the occupancy of sumoylated proteins at a variety of genes by chromatin immunoprecipitation (ChIP) using an antibody that recognizes the yeast SUMO peptide. Surprisingly, we detected sumoylated proteins at all constitutively transcribed genes tested but not at repressed genes. Ubc9, the SUMO conjugation enzyme, was not present on these genes, but its inactivation reduced SUMO at the constitutive promoters and modestly decreased RNA polymerase II levels. In contrast, activation of the inducible GAL1, STL1, and ARG1 genes caused not only a striking accumulation of SUMO at all three promoter regions, but also recruitment of Ubc9, indicating that gene activation involves sumoylation of promoter-bound factors. However, Ubc9 inactivation, while reducing sumoylation at the induced promoters, paradoxically resulted in increased transcription. Providing an explanation for this, the reduced sumoylation impaired the cells ability to appropriately shut off transcription of the induced ARG1 gene, indicating that SUMO can facilitate transcriptional silencing. Our findings thus establish unexpected roles for sumoylation in both constitutive and activated transcription, and provide a novel mechanism for regulating gene expression.

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.

Sumoylation of transcription factor Gcn4 facilitates its Srb10-mediated clearance from promoters in yeast

The small ubiquitin-related modifier (SUMO) is a conserved factor that post-translationally regulates proteins involved in many cellular processes, including gene transcription. We previously demonstrated that promoter-bound factors become sumoylated during activation of inducible genes in yeast, but the identity of these factors, and the role of sumoylation in their function, was unknown. Here we show that the transcriptional activator Gcn4 is sumoylated on two specific lysine residues and in a manner that depends on its ability to bind DNA, indicating that sumoylation occurs after Gcn4 binding to target promoters. Importantly, this functions to facilitate the subsequent removal of the activator from these promoters after recruitment of RNA polymerase II, which can prevent inappropriate transcription of target genes. Furthermore, we show that clearance of sumoylated Gcn4 requires the protein kinase and Mediator complex subunit Srb10, linking activator removal with target gene transcription. Our study demonstrates an unexpected role for protein sumoylation in the process of transcriptional activation.

Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

ABSTRACT The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

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.

Sumoylation controls the timing of Tup1-mediated transcriptional deactivation

The small ubiquitin-like modifier (SUMO) is implicated in various cellular activities, including transcriptional regulation. We previously showed that the yeast activator Gcn4 becomes sumoylated during activation, facilitating its eventual promoter eviction and transcriptional shut off. Here we show that the corepressor Tup1 is sumoylated, at two specific lysines, under various stress conditions. Mutation of these sites has no effect on Tup1 recruitment or RNAP II promoter occupancy immediately following induction. However, Tup1 levels subsequently decrease, while RNAP II and transcription increase in Tup1 mutant cells. Consistent with this, a Tup1 mutant displaying increased sumoylation led to reduced transcription. We also show that coordinated sumoylation of Gcn4 and Tup1 enhances Gcn4 promoter eviction, and that multiple Tup1-interacting proteins become sumoylated after stress. Together, our studies provide evidence that coordinated sumoylation of Gcn4, Tup1, and likely other factors, dampens activated transcription by stabilizing Tup1 binding and stimulating Gcn4 and RNAP II removal.