Proteogenomic analysis reveals alternative splicing and translation as part of the abscisic acid response in Arabidopsis seedlings

Abstract

In eukaryotes, mechanisms such as alternative splicing (AS) and alternative translation initiation (ATI) contribute to organismal protein diversity. Specifically, splicing factors play crucial roles in responses to environment and development cues; however, the underlying mechanisms are not well investigated in plants. Here, we report the parallel employment of short-read RNA sequencing, single molecule long-read sequencing and proteomic identification to unravel AS isoforms and previously unannotated proteins in response to abscisic acid (ABA) treatment. Combining the data from the two sequencing methods, approximately 83.4% of intron-containing genes were alternatively spliced. Two AS types, which are referred to as alternative first exon (AFE) and alternative last exon (ALE), were more abundant than intron retention (IR); however, by contrast to AS events detected under normal conditions, differentially expressed AS isoforms were more likely to be translated. ABA extensively affects the AS pattern, indicated by the increasing number of non-conventional splicing sites. This work also identified thousands of unannotated peptides and proteins by ATI based on mass spectrometry and a virtual peptide library deduced from both strands of coding regions within the Arabidopsis genome. The results enhance our understanding of AS and alternative translation mechanisms under normal conditions, and in response to ABA treatment.

Alternative splicing and translation play important roles in parallel with transcriptional regulation during rice hypoxic germination

“…Rice seed total RNA were ground in liquid nitrogen and extracted using a Plant RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. RNA-sequencing (RNA-seq) experiments were conducted as previously described with minor modifications (Zhu et al, 2017). The resulting cDNA library constructed from rice seed RNA samples (Air_6 h and Hypoxia_6 h) were used for paired end (2 × 125 bp) sequencing on an Illumina HiSeq 4000 platform by Annoroad Gene Technology Co. Ltd. (Beijing, China).…”

“…The rice (Nipponbare) reference genome annotation file (Oryza_sativa.IRGSP-1.0.32) was downloaded from the Ensembl website (http://www.ensembl.org/index.html). Clean reads mapping and subsequent bioinformatic analysis was as described previously (Zhu et al, 2017). The analytical pipeline is summarized in Supplemental Fig.…”

“…In this study, a parallel RNA seq and proteomic approach defined as proteogenomic has been applied to achieve integrative analysis using both transcriptome and proteome data. Given our previous experience in ABA-regulated AS analysis (Zhu et al, 2017), we further improved our analytical pipeline for the determination of AS- and ATI-induced genome coding ability. The results from this study further expand our understanding of genome coding ability in rice seeds, suggesting an underlying regulatory network resulting from AS and ATI during rice hypoxic germination.…”

Integration of large-scale multi-omic datasets: A protein-centric view

“…As standard RNA-seq practices are insufficient for the delineation of many mRNA isoforms, the incorporation of expressed peptide information is crucial in disambiguating this aspect of gene expression. A recent integrative study in Arabidopsis combined short-read RNA sequencing and single molecule long-read sequencing with proteomics and found that 84% of the intron-containing genes were alternatively spliced, primarily through alternative first or last exons, as detected at the RNA level [37*]. The study went further to add another dimension to the analysis: that of differential splicing in cells responding to a stimulus.…”

DRT111/SFPS splicing factor controls ABA sensitivity in Arabidopsis seed development and germination

“…In plants, this mechanism is highly induced in response to external stimuli. Recent studies reported an emerging link between splicing and ABA signalling (Zhu et al, 2017; Laloum et al, 2018). For example, the transcript encoding type 2C phosphatase HYPERSENSITIVE TO ABA 1 (HAB1), a negative regulator of ABA signalling, undergoes alternative splicing.…”