Conservation of an RNA regulatory map between Drosophila and mammals

Abstract

Alternative splicing is generally controlled by proteins that bind directly to regulatory sequence elements and either activate or repress splicing of adjacent splice sites in a target pre-mRNA. Here, we have combined RNAi and mRNA-seq to identify exons that are regulated by Pasilla (PS), the Drosophila melanogaster ortholog of mammalian NOVA1 and NOVA2. We identified 405 splicing events in 323 genes that are significantly affected upon depletion of ps, many of which were annotated as being constitutively spliced. The sequence regions upstream and within PS-repressed exons and downstream from PS-activated exons are enriched for YCAY repeats, and these are consistent with the location of these motifs near NOVA-regulated exons in mammals. Thus, the RNA regulatory map of PS and NOVA1/2 is highly conserved between insects and mammals despite the fact that the target gene orthologs regulated by PS and NOVA1/2 are almost entirely nonoverlapping. This observation suggests that the regulatory codes of individual RNA binding proteins may be nearly immutable, yet the regulatory modules controlled by these proteins are highly evolvable.

Figure 1.

Experimental and analytical approach. (A) S2-DRSC cells were either untreated or treated with two 20-μg doses of dsRNA. After a total of 4 d of incubation with the dsRNA, total RNA was isolated and used for preparing either single-end or paired-end RNA-seq libraries. The single-end libraries were sequenced using 37–45 cycles, while the paired-end libraries were sequenced using 37 cycles for each read. The single-end sequences were trimmed from the 3′ end to a total length of 37 nt prior to alignment. (B) Sequence analysis involved aligning all reads to a combined database of the genome and splice junctions using Bowtie (Langmead et al. 2009). The paired-end alignments were further analyzed using Spliced Paired-End Aligner (SPA) to identify mate pairs that map to the same chromosome, oriented toward one another and within 200 kb of one another. The aligned reads were then analyzed using exonhitter.pl (McManus et al. 2010) to count the number of reads that mapped to exons, splice junctions, and exon–intron boundaries. The read counts were then further analyzed using juncBASE to identify alternative splicing events that were significantly different between the Untreated and ps(RNAi) samples.

Figure 2.

405 PS-regulated pre-mRNA processing events. (Black boxes) Constitutive regions; (white boxes) alternative regions. (Red lines) Splice junctions for the inclusion isoform; (blue lines) junctions for the exclusion isoform. (Red bars) Exonic reads that support the inclusion isoforms; (blue bars) exonic reads that support the exclusion isoforms; (red bars with a black line) reads that support the inclusion isoform, but have a shared portion with the exclusion isoform. Thinner portions of the boxes in alternative first exons and alternative last exons correspond to UTRs.

Figure 3.

Examples of PS-regulated splicing events. Alternative splicing events in the sesB/Ant2 (A), bmm (B), and dre4 (C) genes were identified from the RNA-seq data and validated by RT-PCR. In each case, the RNA-seq coverage and splicing patterns for both the untreated (black) and ps(RNAi) (red) samples are shown along with the annotated transcript models. The RT-PCR validation assays were performed in biological duplicates for both the untreated (lanes 1,2) and ps(RNAi) samples (lanes 3,4). The number of read counts supporting each splicing event in each sample is indicated in the tables on the right.

Figure 4.

A Pasilla RNA-map. (A) Each position in the graph represents the average conserved YCAY cluster score, within a centered sequence window of 45 nt. The conserved YCAY cluster score was calculated for cassette exons that are activated by PS, repressed by PS, and unaffected cassette exons (Fisher's exact test, Benjamini-Hochberg adjusted P-value ≥ 0.95). Only regions adjacent to introns >400 nt were used for scoring. Positions with enriched YCAY cluster scores are indicated by an asterisk (Wilcoxon-rank sum test, uncorrected P-value < 0.01). (B) Positions near cassette exon events, alternative 5′ splice site events, alternative 3′ splice site events, and retained intron events with an enrichment of YCAY clusters. Gray spheres indicate the relative positions containing enriched binding sites. Detailed plots of average conserved YCAY cluster scores are shown in Supplemental Figure 9.