Alternative splicing at the intersection of biological timing, development, and stress responses

Abstract

High-throughput sequencing for transcript profiling in plants has revealed that alternative splicing (AS) affects a much higher proportion of the transcriptome than was previously assumed. AS is involved in most plant processes and is particularly prevalent in plants exposed to environmental stress. The identification of mutations in predicted splicing factors and spliceosomal proteins that affect cell fate, the circadian clock, plant defense, and tolerance/sensitivity to abiotic stress all point to a fundamental role of splicing/AS in plant growth, development, and responses to external cues. Splicing factors affect the AS of multiple downstream target genes, thereby transferring signals to alter gene expression via splicing factor/AS networks. The last two to three years have seen an ever-increasing number of examples of functional AS. At a time when the identification of AS in individual genes and at a global level is exploding, this review aims to bring together such examples to illustrate the extent and importance of AS, which are not always obvious from individual publications. It also aims to ensure that plant scientists are aware that AS is likely to occur in the genes that they study and that dynamic changes in AS and its consequences need to be considered routinely.

Figure 1.

Splicing Signals, SFs, and Spliceosomal Components Involved in Pre-mRNA Splicing. Pre-mRNAs contain intronic splicing signals (splice sites, polypyrimidine tract, and branch point sequences) as well as exonic and intronic splicing enhancer and suppressor sequences (symbols), which are binding sites for SFs. SFs bind to target sequences and help to recruit spliceosomal factors (e.g., U2AF and U1snRNP) to define splice sites and determine spliceosome assembly. Factors associated with pre-mRNA splicing in Arabidopsis are indicated approximately at the steps where they are proposed to act, based on homology to human spliceosome components (Agafonov et al., 2011). Boxes correspond to exons; thin lines correspond to introns.

Figure 2.

Dynamic Regulation of RNA and Protein Expression by AS. Environmental and developmental cues impact gene expression at the level of transcription and AS. Signaling cascades impact the transcription, activity, or subcellular localization of transcription factors and/or SFs. SFs themselves often undergo auto- or cross-regulation by AS. An intimate connection between transcription and AS emerges, where transcription can affect expression levels of SFs and AS of genes dependent on the rate of transcription of RNA polymerase II (Luco et al., 2011); AS can influence the level of transcription factor expression via AS/NMD and the domain composition of transcription factors. Downstream genes may be regulated only at the transcriptional level or the AS level or at both levels.

Figure 3.

Phenotypic Consequences of Aberrant Splicing. (A) to (C) Human mutations affecting splicing. (A) Hutchinson-Gilford Progeria Syndrome leads to premature aging. Mutation of nuclear lamin A, which affects splicing, causes aberrant cell nuclei (C) compared with the regular shape of nuclei in healthy individuals (B). (D) and (E) Plant mutations affecting splicing. (D) An SNP at the wxyb 5′splice site of intron 1 causes aberrant splicing of granule-bound starch synthase resulting in lower levels of amylose and “sticky” rice (left). (E) Phenotypes (left) and corresponding transcript structures (right) of AP3 in Arabidopsis wild type (wt) and the ap3-1 mutant. The ap3-1 mutant contains a point mutation at the 5′splice site of intron 5, leading to skipping of exon 5 and a nonfunctional AP3 protein. The suppressor mutant ap3-11 has a mutation in intron 4 that creates a novel branch point sequence allowing exon 5 to once more be spliced into the mRNA (Yi and Jack, 1998). ([A] to [C] are reprinted from Scaffidi et al. [2005], Figure 1; [D] and [E] are reprinted from Yi and Jack [1998], Figure 1.)

Figure 4.

AS of R Genes during Pathogen Infection. (A) Scheme of the TMV resistance gene N and the two AS isoforms N_S encoding the functional protein and N_L. Inclusion of a 70-nucleotide exon introduces a PTC, leading to a truncated protein variant, based on Dinesh-Kumar and Baker (2000). (B) Scheme of Arabidopsis RPS4 and AS isoforms, based on Zhang and Gassmann (2007). The RPS4 transcript isoform, retaining both introns 2 and 3, was identified in a global transcriptome analysis by Marquez et al. (2012). (C) Scheme of Arabidopsis RPS6 and AS isoforms, based on Kim et al. (2009). The RPS6 transcript isoform retaining intron 3 was identified by Marquez et al. (2012). Note also that The Arabidopsis Information Resource gene model has five exons in the 3′UTR, suggesting that this model (FS) is not in fact the fully spliced model. (D) Scheme of Arabidopsis SNC1 and AS isoforms, based on Xu et al. (2012b). Note that all SNC transcript variants identified by Marquez et al. (2012) retain intron 5.

Figure 5.

Natural Variation in AS. (A) Fruit ripening of strawberry (Fragaria × ananassa). (B) AS of Fragaria × ananassa polygalacturonidase (FaPG) causes a frame shift and a PTC, leading to a nonfunctional protein variant. In soft varieties, the AS isoform Fa-PG1 encoding functional polygalacturonidase predominates, whereas in firm varieties, the AS isoform Fa-PG1-TG corresponding to the truncated, nonfunctional protein predominates (Villarreal et al., 2008).