ABSTRACT
Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, the relative abundance of transcript variants differs across sample types. Differential alternative splicing is common in animal systems, but its extent and significance is not as well known in plants. We examined differential alternative splicing in rice seedlings using RNA-Seq data that included approximately 40 million sequence alignments per library, three libraries per sample type, and four sample types: roots and shoots plus and minus treatment with exogenous cytokinin, a versatile plant hormone that controls a myriad of developmental and stress-related processes in plants. Comparing read alignments to gene model annotations found that for 77% of alternative splicing events proposed in the gene models, each splicing choice was supported by at least one RNA-Seq read alignment. Most genes annotated as alternatively spliced favored one dominant isoform. Of splicing choices where there was abundant support for minor forms, most alternative splicing events affected protein-coding sequence.
Statistical testing of read count proportions identified 90 genes as differentially spliced between rice roots and shoots. By contrast, only four genes were detected as differentially spliced in response to cytokinin. Ten differential splicing events were selected for validation via capillary gel electrophoresis analysis of reverse transcriptase-PCR products, using newly prepared samples from an independent experiment. In nine of ten cases, differential splicing between tissue types was confirmed. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved regions in the encoded proteins, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice.