Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster

Abstract

Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster, expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis-element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215^C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate.

Keywords: 3′READS; Drosophila; RNA polymerase II; alternative polyadenylation; transcription elongation rate.

FIGURE 1.

PASs in the Drosophila melanogaster genome. (A) Experimental design. RNA isolated from Drosophila head or body was subjected to 3′ region extraction and deep sequencing (3′READS) analysis for identification of PAS and analysis of APA isoform abundance. (B) Distribution of PASs and PAS reads in the Drosophila genome. (C) Extension of annotated 3′ ends by 3′READS data (see Materials and Methods for details). A total of 16,908 PASs were found to be in the extended region. The median extension size is indicated. (D) Nucleotide profile around fly PASs. (E) Top 10 enriched 6-mers around the fly PAS. Four regions around the PAS were analyzed, as indicated. Numbers are enrichment scores based on comparison of observed sequences with expected sequences (see Materials and Methods for details). (F) Comparison of 4-mer enrichment between mouse and fly in four regions surrounding the PAS. Values are enrichment score as in E. Several top 4-mers in each graph are highlighted to indicate consistency or distinction between PAS cis-elements in fly and mouse.

FIGURE 2.

APA in Drosophila melanogaster. (A) Percentage of protein-coding genes in the fly genome found to express APA mRNA isoforms using isoform expression cutoffs. At 5% relative abundance cutoff, 78% of fly mRNA genes displayed APA with 4.1 PASs per gene. (B) (Top) Scheme of the different APA types. APA sites are divided into two groups, i.e., 3′-most exon and upstream region (UR). (Bottom) Percentage of genes with APA sites in upstream regions (UR) and/or 3′-most exons. Only genes with APA sites were included. (C) mRNA regions affected by APA. Genes were divided into multiexon or single exon groups. The former was further divided into upstream exon (non-3′-most), intron, and 3′-most exon groups. The number of PASs in each group is indicated. mRNA regions were separated into 5′UTR, coding sequence (CDS), and 3′UTR. For intronic PASs, the mRNA region affected was defined by the exon immediately upstream of the PAS. (D) The 3′UTR size of transcripts from genes without APA sites (single 3′UTR) or with APA sites (shortest and longest isoforms are shown).

FIGURE 3.

3′UTR-APA difference between Drosophila melanogaster body and head. (A) Scheme showing 3′UTR-APA and its analysis. Top two most abundant PAS isoforms per gene were selected for comparison, which are named proximal PAS (pPAS) and distal PAS (dPAS) isoforms, respectively. The distance between the two PASs is considered alternative 3′UTR (aUTR). (B) Scatterplot showing pPAS and dPAS isoform abundance differences between head and body. Two biological replicates were used. Genes with significantly (FDR < 0.05, DEXseq analysis) higher abundance of pPAS isoforms in the body vs. head are shown in blue (1609 genes), and those with higher abundance of dPAS isoforms are in red (234 genes). Total numbers of blue and red genes are shown. (C) Box plot of the 3′UTR length for genes with expression in body and head samples. The weighted mean based on multiple APA isoforms was used to calculate the 3′UTR size of expressed transcripts of each gene. The median value is indicated. (D) UCSC snapshot of 3′READS data for IA-2, showing that pPASs are preferentially expressed in fly bodies while dPASs are in heads. Two replicates are shown. (E) Relationship between the extent of 3′UTR-APA difference and aUTR size. Expressed genes with 3′UTR-APA were evenly divided into five bins based on the aUTR size (distance between pPAS and dPAS), resulting in ∼1300 genes in each bin. The aUTR size range for each bin is shown in the table next to the graph. The extent of 3′UTR-APA difference is represented by relative expression difference (RED), which is the difference in log₂(ratio) of dPAS isoform abundance to pPAS isoform abundance between body and head. Error bars are SEM. Values for genes in bin #1 were compared with those in bin #5 by the Wilcoxon rank sum test, and the P-value is shown. Only PASs with ≥5 reads were used for analysis. (F) Top enriched 6-mers in four regions around the PASs up-regulated in body (top) or in head (bottom). Values are −log₁₀(P), where P is based on the Fisher's exact test.

FIGURE 4.

Upstream region APA difference between fly body and head. (A) Scheme showing various upstream region (UR)-APA isoforms. (B) Scatter plot comparing abundance of UR-APA isoforms and 3′-most exon APA isoform in body vs. head. Genes with significantly higher abundance of UR-APA isoforms in body compared to head are shown in blue (745 genes). Those with higher abundance of 3′-most exon PAS isoforms in the body vs. head are shown in red (212 genes). Two biological replicates were used. Significance of APA was based on the DEXseq analysis (FDR < 0.05). (C) UCSC snapshot of 3′READS data for Dh31, showing that an UR-PAS isoform is preferentially expressed in bodies while 3′-most exon PAS expression is biased to heads. (D) Introns were divided into first (+1), second (+2), last (−1), second to last (−2), and middle (between +2 and −2 introns) groups. Only genes with ≥4 introns were analyzed, and only PASs with ≥5 reads were selected. Expression changes are log₂(ratio) of PAS reads in test sample vs. control sample. Values for five intron groups were normalized by mean-centering. Error bars are SEM.

FIGURE 5.

3′UTR-APA regulation in a fly mutant with a slower RNAPII elongation. (A) Scatter plots comparing 3′UTR-APA isoform abundance between wild-type (WT; w¹¹¹⁸) and mutant (MT; RpII215) in body (left) and head (right), as in Figure 3B. (B) Venn diagram comparing 3′UTR-APA changes between WT and MT in body vs. in head. (C) UCSC snapshot of 3′READS for IA-2, showing that dPAS expression is decreased in mutant body in comparison to the wild-type body. Two replicates are shown. (D) Ratio of dPAS/coding relative mRNA expression levels for IA-2 in wild-type (WT) and mutant (MT) bodies and heads, quantified by RT-qPCR. Data show the mean ± SD normalized to WT body, for at least three independent experiments. Comparisons were performed against WT body and head using an unpaired two-tailed t-test ([*] P < 0.01). (E) Relationship between the extent of 3′UTR-APA difference (relative expression difference, or RED) and aUTR size, as in Figure 3E.

FIGURE 6.

UR-APA regulation in a fly mutant with slower RNAPII elongation. (A) Scatterplot comparing UR-APA and 3′-most exon APA isoform abundance between wild-type (WT) and mutant (MT) in body (left) and head (right), as in Figure 4B. (B) Venn diagram comparing UR-APA changes between WT and MT in body vs. head. Two biological replicates were used for the analysis. (C) Expression difference in intronic APA isoforms between body and head in different intron groups, as in Figure 4D.