Features of conserved alternative splicing events
In this study we identified 537 conserved (CP) AS events in 485 genes. Alternative donor and acceptor events are significantly overrepresented among conserved events, whereas intron retention and exon skipping events are underrepresented. AltA might be the most overrepresented type of AS among the conserved events, because the largest number of AltA events are 3 bp, which would not disrupt the reading frame if proteins are produced from the AS transcripts (Fig. S3; also seen in Campbell et al., 2006). It is possible that there is an overabundance of AltA and AltD types among the conserved events, because the large majority are short (Table 3) and have smaller effects on the coding region, as long as they do not introduce a frameshift. Thus the protein structure and stability are more likely to be conserved if those events are translated. Moreover, short AS located in a domain that do not create a frameshift could be a means of protein regulation (domain switch on/off) without affecting the protein structure. AS events that disrupt protein structure and function would likely be selected against and are less likely to be evolutionarily conserved. We found that conserved AS events are significantly shorter and less likely to be in the 3′UTR than are NEC events. Conserved AS events may be significantly shorter because some of them might have a smaller effect on the protein sequence, if translated, than longer events. In many cases, shorter events might have a less negative impact on protein structure if the transcripts are translated.
We found that conserved AS events are enriched for genes whose products function in the chloroplast. It is possible that AS plays an important role in the regulation of nuclear genes for chloroplast proteins, or that it is involved in producing alternative isoforms of many chloroplast proteins. An AS event modified a functional domain for about half of the genes with conserved events. Thus, if the transcripts are translated, the resulting proteins might have functional changes or might be nonfunctional, depending on the effect of the functional domain modification.
Evolutionary conservation of alternative splicing in angiosperms
In this study we have identified the largest number of conserved AS events between two plant species to date. Both Brassica and Arabidopsis are in the same family (Brassicaceae), whereas some of the previous large-scale comparisons have been between the much more distantly related plants Arabidopsis and rice (Wang & Brendel, 2006; Baek et al., 2008; Severing et al., 2009). Thus one would expect to find a much higher number of conserved events between Arabidopsis and Brassica than between Arabidopsis and rice. However, some previous studies have compared AS event conservation between species within the same family, including three legume species (Medicago, Glycine, and Lotus in Wang et al., 2008) and rice and maize (Severing et al., 2009), but only a small number of conserved events were found. One possibility to explain those results is that the number of ESTs available from the legume species was relatively small, compared with Brassica, Arabidopsis and rice.
Another factor that affects the detected amount of AS conservation is whether conservation of a specific AS event (i.e. the same type of event affecting the same intron in the same position) is compared or whether finding the same type of event in homologous genes between species, even if it affects different introns, is compared. In this study we compared events of the same type affecting the same intron in the same position (conserved position), because these represent true evolutionary conservation of an AS event, as well as events of the same type that affect the same exon–intron junction (conserved junction). We did not analyze events of different types at the same exon–intron junction. Genes that have different types of conserved AS events that affect the same intron, or conserved AS events that affect different introns, might show effects of the AS event that are similar in some cases. For example, if homologous genes from two species both have an IR event, but it affects a different intron and both IR events create a PTC, the effect might be similar.
A drawback of the previous studies of AS conservation in angiosperms and the current study, each of which used Sanger-sequenced ESTs as a source of transcribed sequences, is that the ESTs are not comprehensive with regard to AS and thus many AS events are not detected. That almost certainly leads to an underestimate, perhaps a major underestimate, of the number of conserved AS events between species. Future studies using transcribed sequences derived from higher-throughput sequencing, such as 454 and paired-end Illumina reads, will be needed to estimate the degree of conservation of AS events between species more accurately.
Potential functional and regulatory importance of conserved AS events
Using cross-species analyses to detect conserved AS events is a way of finding AS-creating isoforms with potentially important function, as compared with AS events with no function or ones created by splicing noise (Boue et al., 2003; Ast, 2004; Sorek et al., 2004; Wang & Brendel, 2006; Reddy, 2007; Severing et al., 2009; Keren et al., 2010). In this way, our finding of evolutionary conservation of 537 AS events between Brassica and Arabidopsis suggests a functional importance of many of those AS events. Such events may be good candidates for functional studies, especially those uncharacterized AS events in genes whose functions (of the major or nonAS isoforms) are known. However, not all conserved events are likely to be functional, particularly if the event is conserved because it is created by a mechanism, such as secondary structure, that promotes AS and is conserved between species without having any effect on regulation or function. Likewise, events that are not evolutionarily conserved are not necessarily nonfunctional, because they could have species-specific functions.
What kinds of functional effects might be present for conserved AS events? At the level of transcriptome diversity, AS can regulate mRNA levels by creating transcripts that are targeted to the NMD pathway for degradation, thus lowering the total level of gene expression (Lewis et al., 2003; Hori & Watanabe, 2005; Arciga-Reyes et al., 2006; Kalyna et al., 2012). Several examples of gene regulation by AS and transcript decay have been reported (reviewed in Reddy, 2007; Barbazuk et al., 2008). For example, in the flowering time gene FCA in A. thaliana that controls the transition from the vegetative to the reproductive phase, there are three AS forms produced, none of which encodes a full-length protein, and AS of FCA limits the amount of FCA protein, both spatially and temporally, to prevent precocious flowering (Macknight et al., 2002). Transcript degradation by AS can also have an autoregulatory function. GRP8 and GRP7 in A. thaliana autoregulate and cross-regulate their own expression by AS and NMD (Staiger et al., 2003; Schöning et al., 2008). In this study we found that AS in GRP8 (AT4G39260) is conserved between Brassica and Arabidopsis (Table S2). Transcripts that contain PTCs, in particular those where the stop codon is > 50 bp upstream of the last exon–exon junction or > 350 bp upstream of the end of the transcript, have been considered to be likely targets for degradation by the NMD machinery (reviewed in Wang & Brendel, 2006; Reddy, 2007). However, a recent study of 270 genes in A. thaliana mutants for the NMD pathway revealed that many predicted NMD targets might not actually be degraded by NMD, with only 11–17% of analyzed putative NMD targets overrepresented when NMD was inhibited (Kalyna et al., 2012). Moreover, they found that many IR events that introduce a PTC do not appear to be degraded by NMD. That study highlights the difficulty in identifying real NMD targets in plant transcriptomes without mutant analyses, because the presence of a PTC does not necessarily indicate that a transcript will be degraded. Some of them might be translated to make proteins with altered functions, especially if functional domains are lost or disrupted. Future studies identifying transcripts that are targets of NMD on a genome-wide scale, using transcriptome sequencing of NMD mutants, will be very useful for showing how many of which types of AS events are involved in NMD.
At the level of proteome diversity, AS can have different effects on proteins, such as changing protein function, altering subcellular localization, or affecting the protein affinity or stability (reviewed in Stamm et al., 2005; Reddy, 2007). Analyses of domains allowed us to determine that for half of the genes containing a conserved AS event, the AS modified a functional domain. Three interesting examples, presented earlier, are BPM1, PsbP, and U2AF65a. We also found some cases of short conserved AS events located in predicted domains that did not disrupt the potential protein. Studies of such short AS events in animals (length < 50 nucleotides) revealed that, in some cases, these short nucleotide insertions or deletions modified the secondary protein structure and could, for example, influence the activity of an enzyme or modify the active site conformation (Oakley et al., 2001; Peneff et al., 2001; Wen et al., 2004).
An emerging class of small proteins, called microProteins (miPs) have been described as dominant-negative regulatory proteins disturbing the formation of functional proteins dimers by forming nonfunctional, homotypic protein complexes with their targets (Staudt & Wenkel, 2011). MiPs could result from an AS event that deletes the catalytic, activation or other functional domain from the protein, but retains the protein binding domain. In these cases, the alternative protein could bind its target but create a nonfunctional dimer. Cases of miPs generated by AS that are functional regulators have been shown in animals (Yang et al., 2000; Laitem et al., 2009). Formation of miPs may be an important function of some AS events in plants, including events with evolutionary conservation. We found conservation of an AS event between Brassica and Arabidopsis for a recently reported miP created by AS, Jas1 (also called Jaz10; AT5G13220; Table S2), that was shown to be conserved among several plants (Chung et al., 2010). In the case of BPM1, reported here, the isoform generated by an exon skipping event could be an miP, if this isoform is translated into protein. It is possible that many of the AS transcripts containing PTCs that are not degraded by NMD might be translated into miPs.
Future studies of conserved alternative splicing events will benefit from genome-wide studies of NMD candidates to identify those that might be involved in the regulation of transcript abundance by NMD. Future studies could examine AS event conservation between species at different phylogenetic depths, as well as extending analyses of AS conservation to a genome-wide scale by using RNA-seq approaches to compare matched tissue-type samples from multiple species.