Intronic alternative splicing regulators identified by comparative genomics in nematodes

Abstract

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high-scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.

Figure 1. Images from the Intronerator Genome Browser Showing Alternatively Spliced Genes

Gene isoforms predicted by the Wormbase Consortium are shown in blue, and WABA homology alignments for C. briggsae to this region of the C. elegans genome are shown in purple. Dark purple indicates a region of WABA high homology, light purple corresponds to low homology, and white indicates no homology between species. Regions of alternatively spliced genes: (A) W01F3.1, (B) ZC477.9, (C) ZK637.8, (D) H24G06.1, and (E) C11D2.6 are shown.

Figure 2. An Intronic Element Regulates let-2 Alternative Splicing

The top of this figure shows the alignment of C. elegans and C. briggsae sequences for the alternatively spliced region of let-2. Blue tracks indicate the splicing for the embryonic (top) and adult (bottom) isoforms. The purple track indicates homology between the C. briggsae and C. elegans genomes as determined by WABA. Dark purple tracks indicate regions of strong homology (>70%). The sequence of the first conserved element of intron 10 is shown. The box indicates the part deleted and replaced with the sequence GAA in the del1.2 splicing reporter construct. The lower left part of the figure shows the results of ³²P RT-PCR reactions with primers specific for the splicing reporter. Products for exon 9- or exon 10-containing messages are indicated. In embryos, only usage of exon 9 is detected for either reporter. At the L4 stage, 34% of the wild-type reporter messages contain exon 10 while del1.2 mutant messages contain only a trace amount of this exon.

Figure 3. Conserved Intronic Elements of unc-52 Contain Putative Regulators of Alternative Splicing

(A) C. elegans and C. briggsae sequence alignment is shown for the alternatively spliced portion of unc-52. Not all spliced isoforms are predicted by Wormbase software (blue); see Figure 4A for observed alternative splicing patterns. PhastCons sequence alignment is shown with WABA-designated conservation in bold. Upper line of sequence is C. elegans; C. briggsae is below. High-scoring conserved motifs identified in our pentamer/hexamer analysis of conserved intronic elements flanking alternatively spliced exons, GCATG, TCTATC, CTATCC, CTATC, and TGCAC are underlined. (B) Diagram of alternative splicing reporter constructs for testing putative cis-regulatory splicing motifs. Part of exon 15 through part of exon 19 of unc-52 was cloned into a GFP/lacZ fusion vector with an unc-54 promoter and nuclear localization sequence suitable for expression in C. elegans. Site-directed mutagenesis of the wild-type substrate was performed in order to test putative cis-splicing regulatory elements. A table of the splicing reporter constructs and their alterations is shown. Asterisks denote highly conserved intronic nucleotides deleted by site-directed mutagenesis. To maintain the intron length, yet remove motifs in question, a reporter was also made in which native sequence was replaced with the reverse complement sequence (shown in bold) and a HindIII site (italics) for diagnostic purposes.

Figure 4. Deletion or Mutation of Top Scoring Pentamers or Hexamers Alters unc-52 Alternative Splicing

(A) Autoradiogram of unc-52 reporter gene alternative splicing. Left side shows ³²P RT-PCR analysis (with BamHI digest) performed on strains carrying each of the reporter constructs described in Figure 3B. The reporter is indicated at top of gel. Table on right shows the relative percentage of each alternatively spliced isoform produced from the indicated in vivo splicing reporter constructs. Arrows point to the corresponding band on the gel. Asterisk denotes a non-specific band resulting from RT-PCR. (B) Graphical summary of unc-52 in vivo splicing reporter assays. Horizontal axis indicates spliced mRNA isoform. Vertical axis represents the relative percentage of each isoform. Color key on figure indicates which bars correspond to which strain. Standard deviation and mean values were calculated based on a minimum of three independent RNA extractions and subsequent RT-PCRs for each reporter.