Arabidopsis normalized cDNA library
Alternative splicing landscape in Arabidopsis
Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect ~150,000 splice junctions derived mostly from typical plant introns, including a large number of U12 introns (2,069). Around 61% of multi-exonic genes are alternatively spliced under normal growth conditions. These values are much higher than previously reported and imply a significant role of AS in Arabidopsis transcriptome complexity. Moreover we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using high resolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (~40%) but many IRs have relatively low read coverage and are less well represented in assembled transcripts. Additionally ~51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore the significance of IR in generating transcript diversity is in general overestimated. IR analysis allowed the identification of a large set of cryptic introns inside annotated exons. Importantly 29% of these cryptic introns are in frame indicating a role in protein diversity. Furthermore we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation.