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Am J Bot. 2012 Feb;99(2):219-31. doi: 10.3732/ajb.1100355. Epub 2012 Jan 30.

Mining Arabidopsis thaliana RNA-seq data with Integrated Genome Browser reveals stress-induced alternative splicing of the putative splicing regulator SR45a.

Author information

1
Department of Bioinformatics and Genomics, North Carolina Research Campus, University of North Carolina at Charlotte, 600 Laureate Way, Kannapolis, North Carolina 28081, USA.

Abstract

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.

PMID:
22291167
DOI:
10.3732/ajb.1100355
[Indexed for MEDLINE]
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