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.
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.
Key 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.