Comparative analysis of alternative polyadenylation in S. cerevisiae and S. pombe

Abstract

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, S. cerevisiae and S. pombe Although >80% of the mRNA genes in each species were found to display APA, S. pombe showed greater 3' UTR size differences among APA isoforms than did S. cerevisiae 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 S. pombe, strong motifs surrounding distal PASs lead to higher abundances of long 3' UTR isoforms than short ones, a feature that is opposite in S. cerevisiae 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 S. pombe coordinates with up-regulation of expression for genes involved in translation during cell proliferation. Using S. pombe 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 S. pombe and S. cerevisiae are largely different: S. pombe has many of the APA features of higher species, and Pab2 in S. pombe has a different role in APA regulation than its mammalian homolog, PABPN1.

Figure 1.

Mapping PASs in S. cerevisiae and S. pombe genomes. (A) Schematic showing the experimental design. (B) Statistics of PAS reads mapped to different regions of S. cerevisiae and S. pombe genomes (left) and unique PAS read locations (right). See Methods for definition of PAS read. (C, top) Schematic showing the difference between heterogeneous cleavage and APA. Heterogeneous cleavage sites are merged to a PAS cluster. (Bottom) Distributions of distances between adjacent CSs in S. cerevisiae (left) and S. pombe (right). Two distribution modes were identified by the maximum expectation method (Methods), with the red line showing the distance between CSs within a PAS cluster and the green line the distance between CSs from different PAS clusters. The crossover point (indicated by arrow) was identified for each plot, which was used to group CSs into PAS clusters. (D) APA frequencies of mRNA genes in S. cerevisiae (left) and S. pombe (right). Different abundance cutoffs were used for calling APA sites, leading to different APA frequencies as indicated. The overall APA frequencies were 84.7% and 82.4% in S. cerevisiae and S. pombe genomes, respectively, when the relative abundance cutoff was set at 5%.

Figure 2.

3′ UTR regulation by APA. (A) Schematic showing 3′ UTR length control by APA. The region between the first PAS and last PAS is called alternative UTR, or aUTR. (B) Number of mRNA genes with 3′ UTR APA sites in S. cerevisiae (5583 genes in total) (left) and S. pombe (4579 genes in total) (right). The percentage of mRNA genes with 3′ UTR APA is 78.5% in S. cerevisiae and 71% in S. pombe. The average number of 3′ UTR PASs per gene is 2.6 in S. cerevisiae and 2.5 in S. pombe. (C) Box plots of 3′ UTR size for genes without APA (single) and for genes with APA (the sizes of shortest and longest 3′ UTR isoforms were plotted). (D) Distance between the first and last PASs of genes with 3′ UTR APA. The average values for S. cerevisiae and S. pombe are indicated. (E) Relative expression levels of APA isoforms. APA isoform types were based on PAS locations, as indicated.

Figure 3.

Sequence motifs around PASs. (A) Nucleotide frequencies around the PASs of S. cerevisiae (top) and S. pombe (bottom). (B) Enriched 6-mers around the PASs of S. cerevisiae (top) and S. pombe (bottom). Enrichment values are Z-scores (Methods). Three regions around the PAS were analyzed separately, as indicated. (C) Summary of significant motifs around the PASs of S. cerevisiae and S. pombe based on 6-mer (B) and 4-mer (Supplemental Fig. S2) results. (D) Enriched 6-mers around the first and last 3′ UTR PASs of genes in S. cerevisiae. Values are −log₁₀(P), where P is based on the Fisher's exact test comparing first and last PAS sets. (E) As in D, except that S. pombe data are shown. (F) Summary of PAS strength versus location based on motif analyses. (G) PhastCons scores of the flanking regions of the first and last 3′ UTR PASs in S. cerevisiae (left) and S. pombe (right).

Figure 4.

Metagene analysis of PASs around gene ends. Sense and antisense PASs around the transcript end site (A), stop codon (B), transcription start site (C), or start codon (D) in S. cerevisiae (left) and S. pombe (right). Significant differences between the two yeast species are highlighted by arrows.

Figure 5.

Alternative polyadenylation changes between cells grown in rich versus minimal media. (A) Scatter plot comparing expression changes of proximal PAS (pPAS) isoform (x-axis) between S. cerevisiae cells grown in rich media (RM) versus minimal media (MM) with that of distal PAS (dPAS) isoform (y-axis). Both pPAS and dPAS are in the 3′ UTR. Genes with shortened 3′ UTRs are highlighted in blue, and those with lengthened 3′ UTRs are in red. Gene numbers for both types are shown, and their ratio (number of blue dots to number of red dots) is indicated. (B) As in A, except that data for S. pombe are shown. (C,D) Top Gene Ontology (GO) terms associated with S. cerevisiae (C) or S. pombe (D) genes with shortened 3′ UTRs or lengthened 3′ UTRs in RM versus MM. (BP) biological process; (CC) cellular component. P-value is based on the Fisher's exact test. (E) Venn diagram comparing the genes with shortened 3′ UTRs and genes with up-regulated expression. The P-value (hypergeometric test) indicates the significance of overlap between two genes sets. The enriched GO terms are shown at the bottom with P-values indicated. (F) Distribution of gene expression changes in cell proliferation versus quiescence, as previously reported, for genes with different 3′ UTR changes as analyzed in B. P-values (K-S test) indicating significance of difference between different gene sets are indicated.

Figure 6.

APA regulation by PA factors. (A) 3′ UTR APA analysis of Pcf11-deficient (P_nmt1-pcf11; left) and pab2-null (pab2Δ; right) strains of S. pombe. Each scatter plot compares expression change of proximal PAS isoform (pPAS, x-axis) between an analyzed strain and control, as well as that of distal PAS isoform (dPAS, y-axis) between the strains. Both pPAS and dPAS are in the 3′ UTR. Genes with shortened 3′ UTRs are highlighted in blue, and those with lengthened 3′ UTRs in red. Gene numbers for both types are shown, and their ratio (number of blue dots to number of red dots) is indicated. Note that P_nmt1-pcf11 was grown in minimal media (MM) and pab2Δ in rich media (RM). (B) Venn diagram comparing significantly regulated PASs in P_nmt1-pcf11 with those in pab2Δ cells. P-value (Fisher's exact test) is based on analysis of commonly regulated PASs. (C) Relationship between aUTR size (distance between proximal and distal PASs) and regulation of APA. The two most abundant 3′ UTR isoforms based on PAS reads were selected from each gene. APA regulation of each gene was based on the difference in log₂(dPAS/pPAS) between an analyzed strain and control. A higher value indicates greater up-regulation of the distal PAS isoform. Genes were divided into five groups based on aUTR size. The average value of each group was plotted. (D) Percentage of PAS reads mapped to intergenic regions in wild type, pab2Δ, and P_nmt1-pcf11 strains. The data on wild type were based on both cells grown in RM and in MM. (E) Metagene analysis of PASs in different strains of S. pombe. Significantly altered PAS peaks are highlighted with red arrows.