Non-EST-based prediction of novel alternatively spliced cassette exons with cell signaling function in Caenorhabditis elegans and human

Abstract

To better understand the complex role that alternative splicing plays in intracellular signaling, it is important to catalog the numerous splice variants involved in signal transduction. Therefore, we developed PASE (Prediction of Alternative Signaling Exons), a computational tool to identify novel alternative cassette exons that code for kinase phosphorylation or signaling protein-binding sites. We first applied PASE to the Caenorhabditis elegans genome. In this organism, our algorithm had an overall specificity of > or =76.4%, including 33 novel cassette exons that we experimentally verified. We then used PASE to analyze the human genome and made 804 predictions, of which 308 were found as alternative exons in the transcript database. We experimentally tested 384 of the remaining unobserved predictions and discovered 26 novel human exons for a total specificity of > or =41.5% in human. By using a test set of known alternatively spliced signaling exons, we determined that the sensitivity of PASE is approximately 70%. GO term analysis revealed that our exon predictions were found in the introns of known signal transduction genes more often than expected by chance, indicating PASE enriches for splice variants that function in signaling pathways. Overall, PASE was able to uncover 59 novel alternative cassette exons in C. elegans and humans through a genome-wide ab initio prediction method that enriches for exons involved in signaling.

Figure 1.

Overview of the PASE algorithm. (1) PASE first scans intronic sequences of RefSeq genes for candidate cassette exons. (2) Predicted exons that introduce frameshifts or premature stop codons are removed. (3) Exons that overlap blocks of highly conserved genomic sequence elements are then selected. (4) Finally, the remaining cassette exons are searched with Scansite motifs to identify candidate-signaling exons.

Figure 2.

Examples of Scansite motifs used in PASE. (A) Akt kinase (also known as Protein Kinase B) is a member of the basophilic serine/threonine-specific protein kinase family that selectively phosphorylates the serine/threonine residue (position 0) of protein sequences resembling the linear motif R-X-R-X-X-S/T. (B) The non-catalytic Src Homology 3 (SH3) domain of the tyrosine kinase Src mediates specific protein–protein interactions by binding to ligands with the linear motif resembling R-X-X-P-X-X-P.

Figure 3.

Semi-nested RT-PCR approach to detect novel exons from a pool of 18 tissue RNA samples. In the first round of PCR, an external forward primer targeted to a 5′ upstream canonical exonic sequence is used with a reverse primer targeted to the predicted exon in question. A 1:100 dilution of this first round reaction is then used as the template for the second round of PCR. The second round PCR then uses an internal forward primer targeted to an exonic region between the external forward primer and the previously used reverse exon primer. As an example, the novel C. elegans exon in gene ZK180.2 is shown with the expected PCR product band sizes (261 bp first round PCR, 156 bp second round PCR). The second round reaction was cloned and sequenced for validation of the prediction (DNA ladder is labeled as λ).

Figure 4.

Novel exons in ERα and LRP1 exhibit tissue-specific expression. Flanking exon primers were used in RT-PCR tests for expression of the constitutive splice junctions, while exon-specific semi-nested primers were used to test for the expression of the novel alternative exon. (A) Expression of the ERα constitutive exon 2–3 splice junction and the novel alternative exon across 18 tissues. The constitutive splice junction is expressed in several tissues, shown as a 234 bp PCR product. The novel exon is shown as a 244 bp PCR product that is exclusively included in breast and liver. (B) Expression of the LRP1 constitutive exon 18–19 splice junction and the novel alternative exon across 18 tissues. The constitutive splice junction is expressed in most tissues, showing a 240 bp PCR product. The novel exon is shown as a 128 bp PCR product that is observed in all tissues except uterus. Abbreviations for lanes: DNA ladder (λ), prostate (Pro), breast (Brt), colon (Col), skin (Ski), stomach (Stm), thymus (Thy), lung (Lun), trachea (Tra), placenta (Pla), no template negative control (−N), and pooled tissue control (+P), brain (Brn), retina (Ret), skeletal Muscle (SM), testis (Tes), kidney (Kid), ovary (Ovy), pancreas (Pan), uterus (Utr) and liver (Liv). λ Lanes have size markers spaced at 50-nt intervals up to 350 nt, then the top two bands are for 500 and 766 nt. The strong band corresponds to 200 nt.