Complexity of the alternative splicing landscape in plants

Abstract

Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.

Figure 1.

Frequency of Common Types of AS Events in Humans and Arabidopsis. Proportion of common types of AS events in human (Keren et al., 2010; Reddy et al., 2012b) and Arabidopsis (Barta et al., 2012; Marquez et al., 2012). [See online article for color version of this figure.]

Figure 2.

Integration of Chromatin Landscape, RNA Structure, and RBP-RNA Interactome into AS to Obtain a Comprehensive View of AS Regulation in Plants. Genome-wide high-throughput sequencing tools that can provide insightful information on chromatin landscape, RNA structure, and RBP-RNA interactome are indicated. Integration of these results into AS will allow understanding of crosstalk between AS and other layers of gene regulation and mechanisms of AS. BS-seq, bisulphite sequencing; MNase-seq, micrococcal-nuclease sequencing; ChIP-seq, chromatin-immunoprecipitation sequencing; SHAPE-seq, selective 2′-hydroxyl acylation analyzed by primer-extension sequencing; dsRNA-seq, double-stranded RNA sequencing; ssRNA-seq, single-stranded RNA sequencing; Frag-seq, fragmentation sequencing; RIP-seq, RNA-immunoprecipitation sequencing; PAR-CLIP, photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation; iCLIP, individual-nucleotide resolution cross-linking and immunoprecipitation; iCLAP, individual-nucleotide resolution UV-cross-linking and affinity purification. Table 1 provides a brief description of these tools. [See online article for color version of this figure.]

Figure 3.

General Pipeline for the Analysis of AS Using High-Throughput Sequencing.

Figure 4.

Current NMD Models. Both authentic and premature stop codons are recognized by translation termination factors eRF1 and eRF3. In the course of canonical termination, interaction of eRF3 with poly(A) binding protein (PABP) leads to the efficient release of a peptide (top). According to the long 3′UTR model (middle), this interaction does not occur when the distance between a stop codon (PTC) and poly(A) is extended. eRF3 then interacts with UPF1, which competes with poly(A) binding protein for binding sites. This perturbs translation termination and results in NMD. In the exon junction complex (EJC) model (bottom), EJC is deposited 20 to 24 nucleotides (nt) 5′ of an exon-exon junction during pre-mRNA splicing (Singh et al., 2012). The EJCs are stripped off from mRNA during the first round of translation. In PTC-containing transcripts, termination of translation more than 50 to 55 nucleotides upstream of an EJC triggers RNA decay through NMD by recruiting UPF3 and its cofactors UPF1 and UPF2 (Schoenberg and Maquat, 2012).

Figure 5.

Coupling of AS to NMD. (A) AS can result in both PTC− and PTC+ transcripts. Both PTC+ and PTC− transcripts can be subjected to NMD (NMD+). Not every PTC+ transcript is targeted by NMD (NMD−). (B) Features of NMD sensitive transcripts: 3′UTRs longer than 350 nucleotides; introns in the 3′UTR with a distance >50 to 55 nucleotides (nt) from the authentic stop codon to the downstream splice junction (ds SJ); PTC more than 50 to 55 nucleotides upstream of a splice junction; uORFs in 5′UTR, including uORFs overlapping with authentic start codon (AUG) of the main ORF. PTCs are shown by yellow stop signs. Blue arrows above transcripts indicate long 3′UTRs that can serve as NMD signals.

Figure 6.

AS Can Modulate miRNA-Mediated Regulation of Gene Expression. (A) Generation of RNA splice variants that either contain or lack target sites for miRNA. SJ, splice junction; ES/CE, exon skipping/cassette exon. (B) Modulation of miRNA levels by regulating the splicing pattern of pre-mRNAs encoding enzymes, such as DCL1, involved in miRNA biogenesis. MBS for miR162 (shown in violet) is split between E12 and E13 of DCL1 pre-mRNA. Only one of the four isoforms generated by AS contains a miRNA target site. In addition, miR838A is encoded in the intron 14 of DCL1. AS events and regions are marked red. DCL1 gene structure is not to scale.

Figure 7.

Potential Mechanisms through Which Developmental Cues and Environmental Signals Regulate AS. Signal-induced changes in messengers such as calcium and reactive oxygen species, which are known regulate transcription of numerous genes (Reddy et al., 2011; Mittler et al., 2012; Smékalová et al., 2013), may also regulate AS through protein kinases and protein phosphatases as the activity of many splicing regulators is modulated by protein phosphorylation and dephosphorylation. In addition, the impact of signal-induced changes in cellular milieu, which are known to occur in plants (Seki et al., 2007; Mullen et al., 2012; Chan et al., 2013), on RNA structure and AS is unexplored in plants. See text under Elucidating Pathways That Connect Signals to AS Regulation for details pertinent to this figure. SRPK, Serine/arginine-rich protein-specific kinase; AFC2, Arabidopsis fus3-complementing cDNA 1 (AFC1) homolog (a LAMMER kinase). [See online article for color version of this figure.]