title: Mining Arabidopsis thaliana RNA-seq data with Integrated Genome Browser reveals stress-induced alternative splicing of the putative splicing regulator SR45a.
abstract: OBJECTIVE
  High-throughput sequencing of cDNA libraries prepared from diverse samples (RNA-seq) can reveal genome-wide changes in alternative splicing. Using RNA-seq data to assess splicing at the level of individual genes requires the ability to visualize read alignments alongside genomic annotations. To meet this need, we added RNA-seq visualization capability to Integrated Genome Browser (IGB), a free desktop genome visualization tool. To illustrate this capability, we present an in-depth analysis of abiotic stresses and their effects on alternative splicing of SR45a (AT1G07350), a putative splicing regulator from Arabidopsis thaliana.
  
  
  METHODS
  cDNA libraries prepared from Arabidopsis plants that were subjected to heat and dehydration stresses were sequenced on an Illumina GAIIx sequencer, yielding more than 511 million high-quality 75-base, single-end sequence reads. Reads were aligned onto the reference genome and visualized in IGB.
  
  
  RESULTS
  Using IGB, we confirmed exon-skipping alternative splicing in SR45a. Exon-skipped variant AT1G07350.1 encodes full-length SR45a protein with intact RS and RNA recognition motifs, while nonskipped variant AT1G07350.2 lacks the C-terminal RS region due to a frameshift in the alternative exon. Heat and drought stresses increased both transcript abundance and the proportion of exon-skipped transcripts encoding the full-length protein. We identified new splice sites and observed frequent intron retention flanking the alternative exon.
  
  
  CONCLUSIONS
  This study underlines the importance of visual inspection of RNA-seq alignments when investigating alternatively spliced genes. We showed that heat and dehydration stresses increase overall abundance of SR45a mRNA while also increasing production of transcripts encoding the full-length SR45a protein relative to other splice variants.
authors.affiliation: Department of Bioinformatics and Genomics, North Carolina Research Campus, University of North Carolina at Charlotte, 600 Laureate Way, Kannapolis, North Carolina 28081, USA.
authors.forename: Alyssa A
authors.initials: AA
authors.lastname: Gulledge
authors.forename: April D
authors.initials: AD
authors.lastname: Roberts
authors.forename: Hiral
authors.initials: H
authors.lastname: Vora
authors.forename: Ketan
authors.initials: K
authors.lastname: Patel
authors.forename: Ann E
authors.initials: AE
authors.lastname: Loraine
mesh_terms: D017398:Alternative Splicing; D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D057225:Data Mining; D030561:Databases, Nucleic Acid; D003681:Dehydration / Q000235:genetics; D055864:Droughts; D005091:Exons; D015723:Gene Library; D017343:Genes, Plant; D023281:Genomics / Q000379:methods; D018869:Heat-Shock Response; D059014:High-Throughput Nucleotide Sequencing; D006358:Hot Temperature; D058977:Molecular Sequence Annotation; D022821:RNA Splice Sites; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D012015:Reference Standards; D016415:Sequence Alignment; D012984:Software
chemical_list: D029681:Arabidopsis Proteins; D022821:RNA Splice Sites; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C569740:SR45a protein, Arabidopsis