Alternative polyadenylation (APA) is a widespread mechanism that generates mRNA isoforms with distinct properties. Here we have systematically mapped and compared cleavage and polyadenylation sites (PASs) in two yeast species, and Although >80% of the mRNA genes in each species were found to display APA, showed greater 3' UTR size differences among APA isoforms than did PASs in different locations of gene are surrounded with distinct sequences in both species and are often associated with motifs involved in the Nrd1-Nab3-Sen1 termination pathway. In strong motifs surrounding distal PASs lead to higher abundances of long 3' UTR isoforms than short ones, a feature that is opposite in Differences in PAS placement between convergent genes lead to starkly different antisense transcript landscapes between budding and fission yeasts. In both species, short 3' UTR isoforms are more likely to be expressed when cells are growing in nutrient-rich media, although different gene groups are affected in each species. Significantly, 3' UTR shortening in coordinates with up-regulation of expression for genes involved in translation during cell proliferation. Using strains deficient for Pcf11 or Pab2, we show that reduced expression of 3'-end processing factors lengthens 3' UTR, with Pcf11 having a more potent effect than Pab2. Taken together, our data indicate that APA mechanisms in and are largely different: has many of the APA features of higher species, and Pab2 in has a different role in APA regulation than its mammalian homolog, PABPN1.

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of repeated YSPTSPS heptapeptides and connects transcription with cotranscriptional events. Threonine-4 (Thr4) of the CTD repeats has been shown to function in histone mRNA 3′-end processing in chicken cells and in transcriptional elongation in human cells. Here, we demonstrate that, in budding yeast, Thr4, although dispensable for growth in rich media, is essential in phosphate-depleted or galactose-containing media. Thr4 is required to maintain repression of phosphateregulated (PHO) genes under normal growth conditions and for full induction of PHO5 and the galactose-induced GAL1 and GAL7 genes. We identify genetic links between Thr4 and the histone variant Htz1 and show that Thr4, as well as the Ino80 chromatin remodeler, is required for activation-associated eviction of Htz1 specifically from promoters of the Thr4-dependent genes. Our study uncovers a connection between transcription and chromatin remodeling linked by Thr4 of the CTD.H2A.Z | SWRc I n eukaryotic cells, RNA polymerase II (RNAP II) transcribes all protein coding genes as well as a number of noncoding RNA genes, including those for most snRNAs and microRNAs. Expression of RNAP II-transcribed genes is highly regulated, ensuring that the timing of production, quantity, and structure of the RNAs generated is appropriate. Much of this regulation occurs on the genes themselves, whose accessibility is limited by nucleosomes that can form densely packed chromatin (reviewed in refs. 1 and 2). A significant amount of regulation, however, targets RNAP II itself. Specifically, the C-terminal domain (CTD) of Rpb1, the largest subunit of RNAP II, is subject to a multitude of phosphorylation and dephosphorylation events that mark different steps of the RNAP II transcription cycle (recently reviewed in refs. 3-5). Although many of these modifications occur generally, some appear to play critical roles in expression of specific genes (6-8). The CTD consists of a tandemly repeated heptapeptide, whose consensus sequence, Tyr , is conserved in yeast, which harbors 26 repeats, and vertebrates, with 52 repeats. The emerging picture places this unusual structure in a highly important role in coordinating transcription by RNAP II with other events in the generation and maturation of RNAs.Since its discovery, numerous studies have focused on the role of the entire CTD and the function of individual amino acids within its repeats. At promoters, the unphosphorylated CTD interacts directly with subunits of the Mediator complex (9), which is required for Mediator's role in stimulating transcription (10). Additionally, early studies determined that the CTD is required for full activation by some promoter-bound activators (11-13) whereas it was later shown that the CTD can mediate the influence of activators on pre-mRNA processing (14-16).Several genome-wide analyses uncovered a pattern of CTD phosphorylation that occurs once RNAP II clears promoters (e.g., refs. 17 and 18). Phosphory...

SUMMARY The RNA polymerase II largest subunit C-terminal domain consists of repeated YSPTSPS heptapeptides. The role of tyrosine-1 (Tyr1) remains incompletely understood, as, for example, mutating all Tyr1 residues to Phe (Y1F) is lethal in vertebrates but a related mutant has only a mild phenotype in S. pombe. Here we show that Y1F substitution in budding yeast resulted in a strong slow-growth phenotype. The Y1F strain was also hypersensitive to several different cellular stresses that involve MAP kinase signaling. These phenotypes were all linked to transcriptional changes, and we also identified genetic and biochemical interactions between Tyr1 and both transcription initiation and termination factors. Further studies uncovered defects related to MAP kinase I (Slt2) pathways, and we provide evidence that Slt2 phosphorylates Tyr1 in vitro and in vivo. Our study has thus identified Slt2 as a Tyr1 kinase, and in doing so provided links between stress response activation and Tyr1 phosphorylation.

The C-terminal domain (CTD) of the RNA polymerase II largest subunit consists of a unique repeated heptad sequence of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. An important function of the CTD is to couple transcription with RNA processing reactions that occur during the initiation, elongation, and termination phases of transcription. During this transcription cycle, the CTD is subject to extensive modification, primarily phosphorylation, on its non-proline residues. Reversible phosphorylation of Ser2 and Ser5 is well known to play important and general functions during transcription in all eukaryotes. More recent studies have enhanced our understanding of Tyr1, Thr4, and Ser7, and what have been previously characterized as unknown or specialized functions for these residues has changed to a more fine-detailed map of transcriptional regulation that highlights similarities as well as significant differences between organisms. Here, we review recent findings on the function and modification of these three residues, which further illustrate the importance of the CTD in precisely modulating gene expression.

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.

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