UV damage regulates alternative polyadenylation of the RPB2 gene in yeast

Abstract

Alternative polyadenylation (APA) is conserved in all eukaryotic cells. Selective use of polyadenylation sites appears to be a highly regulated process and contributes to human pathogenesis. In this article we report that the yeast RPB2 gene is alternatively polyadenylated, producing two mRNAs with different lengths of 3′UTR. In normally growing wild-type cells, polyadenylation preferentially uses the promoter-proximal poly(A) site. After UV damage transcription of RPB2 is initially inhibited. As transcription recovers, the promoter-distal poly(A) site is preferentially used instead, producing more of a longer form of RPB2 mRNA. We show that the relative increase in the long RPB2 mRNA is not caused by increased mRNA stability, supporting the preferential usage of the distal poly(A) site during transcription recovery. We demonstrate that the 3′UTR of RPB2 is sufficient for this UV-induced regulation of APA. We present evidence that while transcription initiation rates do not seem to influence selection of the poly(A) sites of RPB2, the rate of transcription elongation is an important determinant.

DNA damage induces targeted, genome-wide variation of poly(A) sites in budding yeast

“…4). With exposure to the UV-mimetic 4NQO, the CPD plot shows a shift to RNAs with longer 39 UTRs, similar to that seen by Yu and Volkert (2013). However, it also reveals that a significant amount of the polyadenylated RPB2 transcripts (;38%) in undamaged cells terminate promiscuously within the coding sequence.…”

“…5B). UV exposure causes damage similar to that of 4NQO, inhibits poly(A) site cleavage in mammalian cells (Kleiman and Manley 2001), and induces a shift to longer transcripts from the yeast RPB2 gene (Yu and Volkert 2013). The 4NQO-induced inhibition of in vitro processing is unlikely to be part of the general environmental stress response as we have shown in previous studies that 39-end formation is not inhibited by heat shock (37°C) for a similar amount of time (Cheng et al 2004;He and Moore 2005;Ghazy et al 2009).…”

“…This technique allowed us to identify patterns of polyadenylation that might otherwise have been missed. The relevance of this novel approach is demonstrated by comparison with a recent study that described a shift to a downstream poly(A) site in the yeast RPB2 gene in response to UV-induced damage (Yu and Volkert 2013). RPB2 has one poly(A) site in the 39 UTR close to the stop codon and a second site that is more distal, and transcripts using both sites are readily detectable in untreated cells by Northern blot analysis (Yu and Volkert 2013) and in our CPD plot (Supplemental Fig.…”

“…It is not currently known how either NMD or NSD responds to DNA damage or, whether under certain conditions, they can act on subsets of their normal targets. There are two published studies showing that stress does not change the relative stability of yeast mRNA isoforms expected to be targets of NMD or NSD, one examining CBP1 transcripts when yeast are grown on glycerol (Sparks et al 1997) and the second looking at RPB2 transcripts in response to UV-induced DNA damage (Yu and Volkert 2013). A whole-genome analysis of changes in mRNA turnover rates will be necessary to resolve the contribution of mRNA stability to changing the ratios of different mRNA isoforms.…”

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion

“…A recent study reported cold shock conditions to globally influence 3 ′ UTR lengths, which regulates gene expression according to the circadian clock (Liu et al 2013). In addition, APA occurs upon UV-induced damage in yeast (Graber et al 2013;Yu and Volkert 2013) and can stress-dependently influence the expression of specific genes, such as HSP70.3, under heat shock conditions (Kraynik et al 2015). Alternative polyadenylation may thus also globally influence mammalian gene expression in conditions of cellular stress.…”