Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe

Abstract

Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3 ′ UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3 ′ RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).

Analysis of RNA Metabolism in Fission Yeast

“…However, it should be noted that the annotations on the main page represent the longest transcripts observed in one study (Rhind et al 2011), which might not correspond to the most common 5 ′ or 3 ′ UTR. 3 ′ termini determined in a study specifically designed to assess the most frequent site(s) of poly(A) tail addition (Mata 2013) are more reliable and generally agree with another study published around the same time (Schlackow et al 2013); these data can be displayed using the Genome Browser on PomBase. For some genes, UTR length changes during a stress response (Leong et al 2014); these data are not currently available on PomBase but could be added in the future.…”

“…Interest in 3 ′ processing of Pol II transcripts in fission yeast has been stimulated by the discovery of widespread alternative polyadenylation using signals that match the mammalian consensus, in contrast to the more degenerate motifs found in budding yeast (Mata 2013;Schlackow et al 2013). Another key development was the demonstration that this process is regulated (McPheeters et al 2009;Yamanaka et al 2010), at least in part by a poly(A) binding protein present in mammals and fission yeast but absent in budding yeast (St-André et al 2010).…”

The Nrd1-like protein Seb1 coordinates cotranscriptional 3′ end processing and polyadenylation site selection

“…S9; data not shown). Our results therefore suggest that the heterogeneity of PAS usage in fission yeast (Mata 2013;Schlackow et al 2013) is not a purely random process but can be modulated by controlling Seb1 levels.…”

“…Recent transcriptome-wide studies indicate that multiple poly(A) sites are used in most eukaryotic genes, as demonstrated in humans (Hoque et al 2013) as well as budding (Ozsolak et al 2010) and fission (Mata 2013;Schlackow et al 2013) yeasts. This process, known as APA, is emerging as a major layer of gene regulation, allowing the inclusion or exclusion of sequences that control the localization, stability, and translation of mRNAs (Tian and Manley 2013).…”

“…For instance, mature snoRNAs are produced from polyadenylated precursors as part of their maturation cycle, which requires the activity of the canonical poly(A) polymerase (Pla1) and the nuclear poly(A)-binding protein Pab2 (Lemay et al 2010). Moreover, recent genome-wide mapping of poly(A) sites in fission yeast revealed that the most prevalent cis elements associated with cleavage site identification are common between mRNA and snoRNA genes (Mata 2013;Schlackow et al 2013). Interestingly, the apparent role of Seb1 in fission yeast 3 ′ end processing mirrors functions of Pcf11 in S. cerevisiae, a protein that also interacts with the CTD of RNAPII (Barilla et al 2001).…”