title: Novel bioinformatics method for identification of genome-wide non-canonical spliced regions using RNA-Seq data.
abstract: METHODS
  During endoplasmic reticulum (ER) stress, the endoribonuclease (RNase) Ire1α 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 Ire1α heterozygous and knockout mouse embryonic fibroblast (MEF) cell lines to identify additional Ire1α targets.
  
  
  RESULTS
  We developed a bioinformatics approach called the Read-Split-Walk (RSW) pipeline, and evaluated it using two Ire1α heterozygous and two Ire1α-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 Ire1α 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.
authors.affiliation: Department of Computational Medicine and Bioinformatics, and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America; Morgridge Institute for Research, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.
authors.forename: Yongsheng
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Justin
authors.initials: J
authors.lastname: Hassler
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Ahdad
authors.initials: A
authors.lastname: Ziyar
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Philip
authors.initials: P
authors.lastname: Li
authors.affiliation: Department of Computational Medicine and Bioinformatics, and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Wright
authors.affiliation: Department of Computational Medicine and Bioinformatics, and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Rajasree
authors.initials: R
authors.lastname: Menon
authors.affiliation: Department of Computational Medicine and Bioinformatics, and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America; Departments of Internal Medicine and Human Genetics, and School of Public Health, University of Michigan, United States of America; Institute for Systems Biology, Seattle, Washington, United States of America.
authors.forename: Gilbert S
authors.initials: GS
authors.lastname: Omenn
authors.affiliation: Department of Computational Medicine and Bioinformatics, and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: James D
authors.initials: JD
authors.lastname: Cavalcoli
authors.affiliation: Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Kaufman
authors.affiliation: Department of Computational Medicine and Bioinformatics, and Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America; Department of Biostatistics, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Maureen A
authors.initials: MA
authors.lastname: Sartor
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics; D059865:Endoplasmic Reticulum Stress; D004722:Endoribonucleases / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D006579:Heterozygote; D006801:Humans; D007438:Introns; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D012326:RNA Splicing; D000071701:Regulatory Factor X Transcription Factors; D012984:Software; D014157:Transcription Factors / Q000235:genetics; D000071717:X-Box Binding Protein 1
chemical_list: D004268:DNA-Binding Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C000605544:Xbp1 protein, mouse; C510185:Ern1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases