Novel Bioinformatics Method for Identification of Genome-Wide Non-Canonical Spliced Regions Using RNA-Seq Data

Setting: During endoplasmic reticulum (ER) stress, the endoribonuclease (RNase) Ire1 alpha initiates removal of a 26 nt region from the mRNA encoding the transcription factor Xbp1 via an unconventional mechanism (atypically within the cytosol). This causes an open reading frame-shift that leads to altered transcriptional regulation of numerous downstream genes in response to ER stress as part of the unfolded protein response (UPR). Strikingly, other examples of targeted, unconventional splicing of short mRNA regions have yet to be reported. Objective: Our goal was to develop an approach to identify non-canonical, possibly very short, splicing regions using RNA-Seq data and apply it to ER stress-induced Ire1 alpha heterozygous and knockout mouse embryonic fibroblast (MEF) cell lines to identify additional Ire1 alpha targets. Results: We developed a bioinformatics approach called the Read-Split-Walk (RSW) pipeline, and evaluated it using two Ire1 alpha heterozygous and two Ire1 alpha-null samples. The 26 nt non-canonical splice site in Xbp1 was detected as the top hit by our RSW pipeline in heterozygous samples but not in the negative control Ire1 alpha knockout samples. We compared the Xbp1 results from our approach with results using the alignment program BWA, Bowtie2, STAR, Exonerate and the Unix "grep" command. We then applied our RSW pipeline to RNA-Seq data from the SKBR3 human breast cancer cell line. RSW reported a large number of non-canonical spliced regions for 108 genes in chromosome 17, which were identified by an independent study. Conclusions: We conclude that our RSW pipeline is a practical approach for identifying non-canonical splice junction sites on a genome-wide level. We demonstrate that our pipeline can detect novel splice sites in RNA-Seq data generated under similar conditions for multiple species, in our case mouse and human.