Mining Arabidopsis thaliana RNA-seq data with Integrated Genome Browser reveals stress-induced alternative splicing of the putative splicing regulator SR45a. American Journal of Botany, February 2012. Gulledge AA, Roberts AD, Vora H, Patel K, Loraine AE.
Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, North Carolina Research Campus.
PREMISE OF THE STUDY:
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.
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.
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.
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.