In Vivo Evidence that Defects in the Transcriptional Elongation Factors RPB2, TFIIS, and SPT5 Enhance Upstream Poly(A) Site Utilization

Abstract

While a number of proteins are involved in elongation processes, the mechanism for action of most of these factors remains unclear primarily because of the lack of suitable in vivo model systems. We identified in yeast several genes that contain internal poly(A) sites whose full-length mRNA formation is reduced by mutations in RNA polymerase II subunit RPB2, elongation factor SPT5, or TFIIS. RPB2 and SPT5 defects also promoted the utilization of upstream poly(A) sites for genes that contain multiple 3 poly(A) signaling sequences, supporting a role for elongation in differential poly(A) site choice. Our data suggest that elongation defects cause increased transcriptional pausing or arrest that results in increased utilization of internal or upstream poly(A) sites. Transcriptional pausing or arrest can therefore be visualized in vivo if a gene contains internal poly(A) sites, allowing biochemical and genetic study of the elongation process.The regulation of eucaryotic gene expression can occur at different and multiple levels. Control of the elongation phase of transcription has been found to be important for a number of genes, most notably those in higher eucaryotes (14,53). It is expected, therefore, that numerous biological controls are in place to ensure that elongation occurs to the extent and degree necessary to result in the proper levels of mRNA for the different genes in the cell. Most importantly, recent evidence has suggested that the elongation process is linked and critical to other posttranscriptional processes such as mRNA capping, splicing, polyadenylation and cleavage, and transport (7,42,48). Elongation can therefore be viewed as the center through which the whole quality control of mRNA formation can be integrated (33,36,42).Recently a number of factors have been identified in yeast and other organisms as either components of the elongating polymerase or as possible modulators of the process of elongation. Biochemical and in vitro studies have demonstrated or suggested a requirement for several of these factors in elongation. As a result of the RNA Pol II structure being recently determined, it is clear that the RPB2 subunit of RNA Pol II may play multiple roles during elongation (16). In vitro evidence confirms a key role for RPB2 because RNA polymerase II (Pol II) containing either the rpb2-10 or rpb2-4 protein fails to elongate well in vitro (41). In particular, the rpb2-10 protein causes arrest at known mammalian pause sites and requires elongation factor TFIIS to overcome the arrest. Moreover, the rpb2-10 allele in combination with a dst1 (encoding TFIIS) deletion causes severe defects in the synthesis of most mRNA in vivo (29) and reduces the induction of a number of other genes (56). These observations suggest that in vivo rpb2-10 actually causes promiscuous arrest of RNA Pol II that requires TFIIS to suppress and overcome.Other important factors involved in elongation are the yeast proteins SPT5 and SPT4, whose higher eucaryotic orthologs are components of the DSIF complex. SPT5 and SPT4...

Interaction between Transcription Elongation Factors and mRNA 3′-End Formation at the Saccharomyces cerevisiae GAL10-GAL7 Locus

“…These results, then, provide support for an in vivo elongation defect for rpb2-10, which increases pausing by pol II in vitro. Our results for spt4, rpb2-10, and rpb2-7 are similar to a recent report from the Denis laboratory (77).…”

“…Frequently, elongation factor mutants are hypersensitive to 6-AU, presumably because they are less tolerant to 6-AU-induced pausing. An rpb2-7 mutant is strongly sensitive to 6-AU but shows phenotypes distinct from rpb1-221 and rpb2-10 (74,76,77). Fig.…”

Ending the message: poly(A) signals then and now

“…In the case of alternative splicing, it is now well known that Pol II processivity set up by specific promoters, by modification of Pol II activity (such as UV-induced hyperphosphorylation of CTD), or by use of an artificial mutant (so-called slow Pol II) can influence alternative splicing patterns (de la Mata et al 2003;Munoz et al 2009). Similarly, loss of elongation factors in yeast has been shown to correlate with increased usage of upstream, cryptic PAS present within genes (Cui and Denis 2003). Very recently, similar mechanisms have been shown to exist for Drosophila APA.…”

Mouse CAF1 Can Function As a Processive Deadenylase/3′–5′-Exonuclease in Vitro but in Yeast the Deadenylase Function of CAF1 Is Not Required for mRNA Poly(A) Removal

“…GAL1 mRNA is polyadenylated at two locations 110 nt apart (25,26). Following the induction of GAL1 mRNA synthesis for 8 min with galactose and repression at time zero with glucose, the rate of deadenylation of the two GAL1 mRNAs was followed in a ycaf1 strain containing LexA-yCAF1, LexA-yCAF1 (S199A/ E201A), or LexA.…”