Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis

Abstract

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 an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using highresolution 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 was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out 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. [Supplemental material is available for this article.]Alternative splicing (AS) is a widespread mechanism which increases transcriptome and proteome complexity and controls developmental programs and responses to the environment in higher eukaryotes. The splicing process, removal of introns and ligation of exons, is performed by a large RNA-protein complex, the spliceosome, consisting of five small nuclear RNAs (snRNAs) and about 180 proteins with different functions (Wahl et al. 2009). Assembly of the spliceosome on introns in a precursor messenger RNA (pre-mRNA) is directed by cis elements and trans-acting factors (Black 2003;Stamm et al. 2005). The cis sequences include the splice sites, branchpoint, and polypyrimidine tract which have degenerate consensus sequences in higher eukaryotes. While many splice sites are selected in all transcripts (constitutive splicing), others are used to various levels, resulting in alternative transcripts. Selection of such alternative splice sites is affected by auxiliary cis elements located within exonic and intronic sequences, termed splicing enhancers and silencers. These elements are binding sites for trans-acting splicing factors, for example, hnRNP and SR proteins. These proteins, in addition to their functions in constitutive splicing, play a key role in AS by inhibition or ...

The Differential Transcription Network between Embryo and Endosperm in the Early Developing Maize Seed

“…Furthermore, alternative 39 splice sites (A3SS; 21.0%) or alternative last exons (ALE; 11.5%) and alternative 59 splice sites (A5SS; 12.2%) or alternative first exons (AFE; 11.7%) were other types of common AS events observed in our data (Fig. 3C), in agreement with recent findings in rice , Arabidopsis (Marquez et al, 2012), human , and Drosophila melanogaster (Daines et al, 2011;Graveley et al, 2011). In addition, a higher frequency of tissueregulated events of each type and more junctions were detected in embryo than in endosperm ( Fig.…”

“…Retained intron, in which a single intron is alternatively included or spliced out of the mature mRNA via an intron-definition splicing mechanism, was the most prevalent type of AS event (30.4%; Fig. 3C), consistent with previous studies in plants (Black, 2003;Wang and Brendel, 2006;Barbazuk et al, 2008;Filichkin et al, 2010;Li et al, 2010;Lu et al, 2010;Zhang et al, 2010;Marquez et al, 2012) but in contrast to animal AS events, where exon skipping (cassette exons or coordinate cassette exons) is the predominant mechanism (Sultan et al, 2008;Wang et al, 2008;Daines et al, 2011;Graveley et al, 2011). Furthermore, alternative 39 splice sites (A3SS; 21.0%) or alternative last exons (ALE; 11.5%) and alternative 59 splice sites (A5SS; 12.2%) or alternative first exons (AFE; 11.7%) were other types of common AS events observed in our data (Fig.…”

Genome‐wide analysis of alternative splicing events in Hordeum vulgare: Highlighting retention of intron‐based splicing and its possible function through network analysis

“…As most of the intron retention events in Arabidopsis happened in PTC+ isoforms, it possibly means that intron retention, as well as the other nonsense-generating splicing events play a significant role in RUST-regulated gene expression. The coupling of AS with NMD appears to be widespread among eukaryotes [49] that their coupling was showed in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, establishing a complex loop in AS regulation ( [50]). …”

ABA signalling is fine-tuned by antagonistic HAB1 variants

“…Alternative splicing (AS) regulates not only transcript levels but also transcript isoforms, giving rise to proteins that differ in subcellular localization, stability and function [23][24][25] . More than 61% of intron-containing Arabidopsis genes undergo AS under normal conditions 26 . The percentage of genes showing AS will dramatically increase when plants are subjected to abiotic stresses.…”