Alternative splicing in plants: directing traffic at the crossroads of adaptation and environmental stress

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ScienceDirect Alternative splicing in plants: directing traffic at the crossroads of adaptation and environmental stress Sergei Filichkin1,2, Henry D Priest3,4, Molly Megraw1,2 and Todd C Mockler1,2,3,4 In recent years, high-throughput sequencing-based analysis of plant transcriptomes has suggested that up to 60% of plant gene loci encode alternatively spliced mature transcripts. These studies have also revealed that alternative splicing in plants can be regulated by cell type, developmental stage, the environment, and the circadian clock. Alternative splicing is coupled to RNA surveillance and processing mechanisms, including nonsense mediated decay. Recently, non-protein-coding transcripts have also been shown to undergo alternative splicing. These discoveries collectively describe a robust system of post-transcriptional regulatory feedback loops which influence RNA abundance. In this review, we summarize recent studies describing the specific roles alternative splicing and RNA surveillance play in plant adaptation to environmental stresses and the regulation of the circadian clock. Addresses 1 Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA 2 Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR 97331, USA 3 Division of Biology and Biomedical Sciences, Washington University, Saint Louis, MO 63130, USA 4 Donald Danforth Plant Science Center, Saint Louis, MO 63132, USA Corresponding authors: Mockler, Todd C ([email protected]) and Filichkin, Sergei ([email protected])

Current Opinion in Plant Biology 2015, 24:125–135 This review comes from a themed issue on Genome studies and molecular genetics Edited by Insuk Lee and Todd C Mockler For a complete overview see the Issue and the Editorial http://dx.doi.org/10.1016/j.pbi.2015.02.008 1369-5266/# 2015 Elsevier Ltd. All rights reserved.

Introduction Alternative splice site selection during eukaryotic precursor-mRNA (pre-mRNA) processing results in the production of multiple mature mRNA isoforms from a single gene locus, known as alternative splicing (AS). AS expands proteomic diversity and regulates gene expression at the post-transcriptional level. Splice site selection has been shown to be regulated by cell type, developmental stage, and cellular stress. High-throughput Sequencing-based www.sciencedirect.com

estimates of alternatively spliced transcripts in Arabidopsis thaliana range from 42% [1] to 61% [2]. It is likely that many more genes will be shown to undergo AS as transcriptomes of plants grown under stress are evaluated and as computational tools used for NGS-based predictions of splice isoforms are improved [3,4,5,6]. Alternatively spliced mRNAs in Arabidopsis can accumulate at substantial levels [1,5,7,8,9]. AS frequently generates nonsense mRNA carrying in-frame premature termination codons (PTCs) [1,2,5,8,9,10]. PTC-harboring (‘PTC+’) mRNAs are, in many cases, rapidly degraded by the cellular nonsense-mediated mRNA decay (NMD) machinery. This type of AS is often referred to as ‘unproductive alternative splicing’ [11,12]. Some PTC+ mRNA escape NMD and produce truncated proteins which may be missing key functional domains. In plants, evidence suggests that stable NMD-insensitive PTC+ mRNAs play important roles in transcriptome adaptation to developmental demands and/or plant responses to environmental stresses [5,9,13]. In this review, we summarize recent findings describing mechanisms of AS, coupling of AS to the RNA surveillance machinery, and the specific roles of AS and NMD in plant adaptation to the environmental stress and in regulation of the circadian clock.

Pre-mRNA splicing machinery Eukaryotic pre-mRNA splicing is mediated by a large ribonucleoprotein (RNP) complex known as the spliceosome. Both constitutive and AS of pre-mRNA is catalyzed by the spliceosome. This large RNP complex is comprised of five small nuclear RNAs, small nuclear ribonucleoproteins (snRNPs), and hundreds of spliceosomal proteins [14–16]. There are strong similarities in exon– intron structure and a significant conservation of splice site consensus signals within pre-mRNAs between plants and animals [17]. Members of the serine/arginine-rich (SR) protein family mediate spliceosomal pre-mRNA binding specificity. The SR proteins have a modular structure consisting of one or two RNA recognition motifs (RRMs) and an arginine/ serine-rich (RS) domain [5,18–20]. The RRM domains are involved in pre-mRNA splice site selection, whereas RS domains likely mediate protein–protein interactions and act as splicing activators [21]. Some SR proteins act as splicing repressors (e.g., dephosphorylated mammalian Current Opinion in Plant Biology 2015, 24:125–135

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SRp38 inhibits splicing during heat shock), whereas other SR proteins antagonize the inhibitory activity of the heterogeneous nuclear RNPs (hnRNPs) to activate splicing [22].

particularly potent NMD response can be triggered by short PTC-harboring exons (present in certain mammalian splicing factors and termed poison cassette exons [12]). In Arabidopsis, HSFA2, RVE2 [24], and SR30 [1] transcripts provide examples of such poison cassette exons.

Coupling of alternative pre-mRNA splicing to NMD

Compatible models of on-demand switches toward unproductive AS via intron retention (IR) proposed for the fern Marsilea vestita [25,26] and Arabidopsis [24] are likely to be broadly relevant for other eukaryotic systems. Studies of masked mRNA storage during development of M. vestita microspores favor the hypothesis that IR mRNA intermediates escape cytoplasmic NMD via sequestration in the nucleus; these intermediates are spliced and released depending on cellular demands. Boothby and Wolniak [25] demonstrated that unspliced and partially spliced pre-mRNAs can be stored in nuclear speckles in

Alternatively spliced mRNAs harboring in-frame PTCs can be recognized by the RNA surveillance machinery as aberrant; these transcripts are targeted by the nonsense-mediated decay (NMD) pathway and rapidly degraded. Key current NMD models are illustrated in Figure 1. Recognition of an aberrant mRNA by NMD is determined by NMD-eliciting transcript features, such as PTC location relative to the initiation codon, length of the 30 untranslated region (30 UTR), and/or presence of short overlapping open reading frames in the 50 UTR [23]. A Figure 1

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Current NMD models. (a) The exon junction complex (EJC) model was proposed for mammalian cells and relies on the position of the PTC relative to the downstream exon/exon junction and associated EJC. The EJC proteins are deposited onto spliced mRNA during pre-mRNA splicing and remain associated with spliced mRNAs during their transport into the cytoplasm. The EJC proteins are removed in the cytoplasm during the pioneer round of ribosome scanning, resulting in an NMD-recalcitrant mRNA. Upon premature translation termination, the downstream EJC recruits UPF factors. The subsequent formation of the EJC/UPF complex determines whether a stop codon is interpreted as being PTC. UPF2 and UPF3 recruit UPF1 to the EJC and cooperatively stimulate both the ATPase and RNA helicase activities of UPF1. NMD is triggered by interaction of the phosphorylated UPF1 protein with the ribosomal release factors (eRF1 and 3) during the pioneer round of translation. (b) The Faux (false) 30 UTR model [55] presumes that NMD is regulated by interactions of a terminating ribosome with the 30 UTR binding protein(s). Normal translation termination occurs only when a terminating ribosome is in close proximity to the 30 UTR that allows interaction between eRF3 and the poly(A) binding protein complex (PABPC) associated with the 30 poly(A) tract. Premature termination occurs when translation terminates distal from the 30 UTR and the terminating ribosome cannot interact with PABPC but instead recruits NMD UPF factors. Plant NMD incorporates some features of both models. However, in contrast to mammalian cells where mRNAs transcribed from intronless genes can escape NMD, plant single exon transcripts harboring a PTC can be detected and degraded by NMD machinery. The cap-binding protein complex (CBC), an important component of the existing models, is not essential for NMD in plants [56]. The PTC position [57], 30 UTR length [58] and the presence of the short upstream ORFs (uORFs) [59] appear to be important NMD-eliciting transcript features [23]. The EJC proteins are conserved across many eukaryotes, however, direct involvement of plant EJC orthologs in plant NMD remains poorly studied. Arabidopsis eIF4A-III (an EJC ‘anchor’ on mammalian mRNAs) and ALY/Ref orthologs co-localize to the nucleolus together with Mago, Y14, and RNPS1/SR45 [60,61]. This finding suggests that the EJC-mRNA complex is at least assembled in plant cell nucleus. Current Opinion in Plant Biology 2015, 24:125–135

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Alternative splicing in plants: adaptation and stress Filichkin et al. 127

association with the exon junction complex protein, Mago nashi. Removal of retained introns from these sequestered intermediates and release of mature mRNAs is developmentally triggered in a spliceosome-dependent fashion [26]. At least 17% of all alternatively spliced multi-exon, protein-coding genes in Arabidopsis produce mRNA isoforms that are targeted by UPF-mediated NMD [27]. Despite harboring multiple PTCs, the majority of Arabidopsis mRNAs retaining full introns escape NMD [2,8,23]. In contrast, many transcripts with partially retained introns are degraded through the NMD pathway [8,23,24,28,29]. The basis and molecular mechanisms of such discrimination remain to be established.

Direct spliceosomal links to environmental adaptation SR proteins are key regulators of plant responses to environmental conditions. Plant responses to environmental stresses are associated with profound changes in global AS patterns. Stress-driven AS strongly affects expression of genes associated with stress response pathways and genes that encode spliceosomal components [1,5,9,24,30–32]. Specific splicing factors modulate selection of alternative splice sites in response to specific types of cellular stress. Pre-mRNAs of the majority of Arabidopsis SR proteins undergo extensive AS induced by various environmental stresses, and hormones such as abscisic acid (ABA), indoleacetic acid (IAA), and 6-benzyl aminopurine (BA) [1,30,33]. By using high-coverage (200) RNA-seq data, Ding et al. [32] demonstrated that high salinity stress frequently introduces PTCs into the mRNAs encoding SR proteins through alternative 50 and 30 splice site selection and IR events. Transcripts of 15 of 18 tested SR genes were alternatively spliced when plants were grown under high salinity conditions. Abiotic stress-driven production of the PTC+ SR mRNAs can shift the ratio of the productive to unproductive isoforms (e.g., SR30 [1]). Profound changes in AS of the SR mRNAs are accompanied by alteration of global AS patterns in 49% of all intron-containing genes [32]. A large proportion of these genes encode proteins involved in biotic/ abiotic stress response pathways. In addition to environmental stresses triggering specific AS events, evidence shows that spliceosomal proteins play crucial roles in the proper function of Arabidopsis stress response pathways. Cruz et al. [34] showed that SR34, SR34b, SCL30a, SCL28, SCL33, RS40, and SR45 SFs regulate ABA signaling pathways. ABA is a hormone that facilitates developmental arrest under environmental stress. Cui et al. [35] reported that the Arabidopsis SmLIKE PROTEIN 5 (LSm5) encoded by the SUPERSENSITIVE TO ABA AND DROUGHT 1 (SAD1) gene increases www.sciencedirect.com

precision of splice site recognition. Mutations in SAD1 result in a genome-wide increase of AS, especially among pre-mRNAs of salt stress-responsive genes [35]. Nuclear cap-binding complex protein subunits CBP20 and CBP80 also affect selection of splice sites [36]. The cbp20 and cbp80 mutations caused numerous changes in AS events in transcripts of genes encoding splicing factors and stress-related proteins. CBP20 and CBP80 are involved in modulation of the salt-stress response [37]. The components of both the spliceosome (SR proteins) and splicing related genes (CBPs and LSm proteins) are required for proper expression of Arabidopsis stress-response pathways. The components of the Arabidopsis splicing machinery implicated directly or indirectly in regulation of stress responses are summarized in Table 1. The role of SR45 in stress responses has been well characterized. SR45 is a member of a conserved family of structurally and functionally related non-snRNPs [5,20,38]. SR45 interacts with U1-70K and U2AF35b spliceosomal proteins and these interactions are thought to assist in formation of a bridge between 50 and 30 splice sites during spliceosome assembly [39]. SR45 and other factors, including SCL33, RSZ21, and RSZ22, interact with U1-70K to regulate both constitutive and AS [40– 43]. A loss-of-function sr45-1 mutant is hypersensitive to ABA [44]. By screening T-DNA insertion lines and using FWA transgene silencing as a reporter system Ausin et al. [45] identified SR45 as a component of the RNAdirected DNA methylation (RdDM) pathway. It is possible that SR45 may affect siRNA production through the splicing of transcripts involved in the RdDM pathway [45]. Arabidopsis spliceosomal factor SNW/Ski-interacting protein (SKIP) has been implicated in regulation of abiotic stress responses such as ABA signaling and confers tolerance to osmotic and high-salt stresses [46]. SKIP physically interacts with SR45 protein and regulates AS of numerous genes including circadian clock genes PRR7 and PRR9 [47]. Mutations in the SKIP gene increase the circadian period in a temperature-sensitive manner [47]. RDM16 is another example of a pre-mRNA splicing factor that is required for the regulation of abiotic stress responses. It is an important component of the RdDM pathway [48]. rdm16 mutants have severe morphological defects and are hypersensitive to salt stress and ABA treatment [48]. RDM16 is likely to be directly involved in RdDM pathway because the rdm16 mutation is not defective in splicing of other tested RdDM genes. Protein arginine methyltransferase 5 (PRMT5, also known as SKB1) affects salt stress-driven pre-mRNA splicing through methylation of an snRNP Sm-like4 Current Opinion in Plant Biology 2015, 24:125–135

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Table 1 The components of the Arabidopsis splicing machinery implicated in regulation of stress responses Gene Pre-mRNA splicing factors SR34 AT1G02840 SERINE/ARGININE-RICH PROTEIN SPLICING FACTOR 34 AT1G09140 SR30 SERINE/ARGININE-RICH PROTEIN 30 SR34b AT4G02430 SERINE/ARGININE-RICH PROTEIN SPLICING FACTOR 34b AT3G13570 SCL30a SC35-LIKE SPLICING FACTOR 30a SCL28 AT5G18810 SC35-LIKE SPLICING FACTOR 28 SCL33 AT1G55310 SC35-LIKE SPLICING FACTOR 33 RS40 AT4G25500 ARGININE/SERINE-RICH SPLICING FACTOR 35 AT1G55310 SCL33 SC35-LIKE SPLICING FACTOR 33 RS2Z33 AT2G37340 ARGININE/SERINE-RICH ZINC KNUCKLE-CONTAINING PROTEIN 33 AT5G52040 RS41 ARGININE/SERINE-RICH SPLICING FACTOR 41 ARGININE/SERINE-RICH SPLICING RS31 FACTOR 31 RS31a ARGININE/SERINE-RICH SPLICING FACTOR 31a RSZ33 AT2G37340 ARGININE/SERINE-RICH ZINC KNUCKLE-CONTAINING PROTEIN 33 AT1G16610 SR45 b ARGININE/SERINE-RICH SPLICING FACTOR 45 AT1G07350 SR45a ARGININE/SERINE RICH-LIKE PROTEIN 45a AT5G48870 LSm5 SUPERSENSITIVE TO ABA AND DROUGHT 1, AT1G28060 RDM16 RNA-DIRECTED DNA METHYLATION 16 SKIP AT1G77180 SNW/SKI-INTERACTING PROTEIN

Other splicing-related proteins CBP20 AT5G44200 CAP-BINDING PROTEIN 20 CBP80 AT2G13540 CAP-BINDING PROTEIN 80 PRMT5 AT4G31120 PROTEIN ARGININE METHYLTRANSFERASE 5 a b c

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Indicated stress treatments affect AS of the pre-mRNA of the listed SF. Affects AS of SR genes including SR30 [38]. Affects AS of genes encoding stress-related proteins (ABA), stomatal ABA signaling, and SR SFs. No direct stress experiments published.

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(LSm4 [49]). prmt5 mutation results in a broad range of RNA splicing defects [50,51]. Zhang et al. showed that PRMT5 associates with chromatin and alters methylation of histone 4 arginine 3 (H4R3sme2) [49]. During salt stress, the level of H4R3sme2 is reduced, resulting in induction of the expression of stress-responsive genes. PRMT5 may regulate the salt-stress response both at the transcriptional level (by altering the histone 4 methylation status) and at the pre-mRNA levels through methylation of LSM4. In addition to abiotic stress response facilitation, mutations in PRMT5 also result in a change of the AS pattern of the core clock gene PRR9 and impair plant circadian rhythms [51].

Linkage between RNA surveillance and abiotic stress responses Arabidopsis NMD-impaired mutants upf1-5 and upf3-1 show pre-activation of certain stress-response networks, including immunity-associated genes, resulting in decreased susceptibility to bacterial pathogens such as P. syringae [24,52]. Microarray profiling of the upf15 transcriptome suggests that the transcripts targeted by the UPF1-dependent NMD pathway include numerous defense-related genes [52]. RNA-seq data also suggests that the transcriptomes of the upf1-5 mutant and heat-stressed plants share overlapping repertoires of the IR events [24]. Current data support the hypothesis that the UPF-dependent RNA-surveillance pathway contributes to control of stress response networks. Moreover, the intron-retaining mRNAs of both UPF1impaired and heat stressed Arabidopsis transcriptomes are enriched in genes involved in splicing and response to stress. Expression of the SR splicing factors themselves is extensively regulated by unproductive AS coupled with the UPF-dependent NMD pathway. Using the upf3 loss-of-function mutant, Palusa and Reddy [53] showed that approximately half of the 53 PTC+ splice variants generated by the AS of 13 Arabidopsis SR genes are NMD-sensitive. AS events control the expression of productive transcripts that encode members of the SR [11,12] and hnRNP [54] families to tune processes to particular cellular demands or perturbations. In plants, an example of such AS/ NMD-linked negative feedback loop is presented by mRNAs of GLYCINE RICH PROTEIN (GRP) 7 and 8 [28].

Environmental adaptation via AS of nonprotein coding RNAs The splicing of non-protein coding RNA transcripts is an emerging area of study, specifically in plants. Two major classes of such RNA molecules are micro-RNA-containing transcripts (miRNAs) and long non-coding RNAs (lncRNAs). www.sciencedirect.com

Two different forms of miRNA regulation via splicing are possible. The first is the splicing of miRNA primary transcripts (pri-miRNAs) in order to control the production of pre-miRNA hairpins. There is accumulating evidence that many plant pri-miRNAs contain introns [62] and have multiple alternatively spliced isoforms [63–65]. It is also apparent that AS of pri-miRNAs can lead to increased or decreased accumulation of mature miRNAs in plants [65–68]. Though the precise causes for this are not yet clear, evidence shows AS of the dicistronic primiR842-miR846 disrupts stem-loop structures and inhibits maturation of miRNAs [69]. Removal of an intron may also facilitate formation of pri-miRNA stem-loop structures necessary for maturation [70]. There is also a possibility that pri-miRNA splicing may facilitate a recruitment of specific RNA binding proteins which are necessary for miRNA processing [62]. The second form of splicing-related miRNA regulation occurs in cases where the miRNAs are located in introns and the host transcript is alternatively spliced in a way that affects miRNA production. The biogenesis of some intronic miRNAs occurs through the canonical pathway that includes cleavage by Drosha [69]. Some intronic miRNAs, known as miRtrons, are not cleaved by Drosha [71]. For these miRtrons the pre-miRNA is released from the transcript by the splicing machinery [72]. Biogenesis of certain plant miRtrons appears to be controlled by stress. Arabidopsis MIR400 provides an example. Heat stress-induced AS of a transcript that contains this miRtron results in decreases in levels of the mature miRNA [73]. Although it remains unclear whether environmental stress is the most common inducer for the regulation of plant miRNA production through AS, recent findings link abiotic stress, AS, and the biogenesis of both miRNAs and miRtrons [69,70]. A recent study in barley [66] demonstrates that heat stress induces the AS of pri-miRNAs 160a and 5175a, which in turn affects accumulation of mature miRNA — although the effect of this AS on cellular stress response is unknown. At least 50% of annotated Arabidopsis lncRNAs contain introns [70]. Production of a substantial proportion of Arabidopsis lncRNAs is regulated by abiotic stress [70] and many undergo AS [74]. Even though the exact roles that AS plays in lncRNA biology remain poorly investigated, a general consensus is that it can affect functional portions of RNA and alter interactions with lncRNA targets. In turn, lncRNAs can regulate AS of pre-mRNAs. Bardou et al. [75] identified two Arabidopsis nuclear speckle RNA-binding proteins (NSRs) that act as nuclear regulators of AS. NSRs are expressed in primary and lateral roots meristems (Figure 2). Both genes are required for Current Opinion in Plant Biology 2015, 24:125–135

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Figure 2

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Long non-coding ASCO-RNA interacts with NSR splicing regulators and diverts them from their alternatively spliced mRNA targets. Such a diversion of NSRs results in alteration of the balance of alternatively and constitutively spliced isoforms of NSR targets and affects number and growth of lateral roots. Auxin and ASCO-RNA play antagonistic roles in the promotion of lateral root development.

normal development of lateral roots because the nsra/nsrb double mutant results in reduction in the numbers and lengths of the lateral roots. NSRa is expressed constitutively, whereas NSRb can be induced by auxin. Consistently, the lateral root growth in nsra/nsrb mutants was less sensitive to auxin [75]. Interestingly, the level of a lncRNA called the AS competitor RNA (ASCO-RNA) is elevated in the nsra/nsrb double mutant, suggesting that NSRs regulate the expression of lncRNAs. The majority of 85 genes that are alternatively spliced after auxin treatment are dependent on NSRs. Bardou et al. [75] also showed that NSRa and NSRb physically interact with their alternatively spliced pre-mRNA targets as well as with ASCO-RNA. ASCO-RNA competes with alternatively spliced NSR-dependent mRNA targets for binding to NSR splicing regulators. Such competitive binding suggests that ASCO-RNA displaces splicing regulators from their mRNA targets. Current Opinion in Plant Biology 2015, 24:125–135

Because 70% of annotated Arabidopsis mRNAs are associated with long noncoding natural antisense transcripts [76], the regulation of AS via direct base pairing of premRNA with lncRNAs may be common in plants. RNA surveillance mechanisms recognize and degrade many plant noncoding transcripts [77,78]. Experiments using whole-genome tiling microarrays and Arabidopsis upf1 and upf3 NMD-impaired mutants suggest that mRNA-like noncoding and natural antisense RNAs are degraded by the UPF-dependent NMD machinery [78]. The role of NMD in regulation of stability and decay of lncRNAs remains poorly understood.

AS plays an important role in the regulation of plant circadian clock and its adaptation to environmental stress The Arabidopsis circadian oscillator presents a particularly interesting regulatory system because abundance and www.sciencedirect.com

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timing of alternatively spliced circadian transcripts can be profoundly modulated by environmental stress. The circadian clock is a time-keeping mechanism that allows eukaryotes to anticipate daily fluctuations of environmental cues such as light, temperature, and even optimal times for pathogen infection. Circadian clocks in higher plants operate through interlocked transcriptional regulatory feedback loops [79]. The central circadian oscillator also orchestrates the timing of expression of numerous gene networks including those that regulate response to biotic and abiotic stresses. Many components of the Arabidopsis central circadian oscillator and/or regulators of clock output are alternatively spliced and produce PTC-harboring isoforms [8,80]. Both biotic and abiotic stress can alter oscillation profiles of the nonsense

isoforms and reversibly shift the ratio of a constitutively spliced isoform to its PTC-containing counterpart. Production of alternatively spliced circadian transcripts is typically synchronized in phase with their functional mRNAs [1,8,24]. Environmental stresses can alter abundance [1,8] and/or desynchronize daily cyclical expression profiles of circadian PTC+ mRNAs relative to their functional counterparts [24]. Examples of circadian NMD-inducing PTC+ transcripts: GRP7 and GRP8 [28], RVE2 [8,24], and specific events in LHY, PRR9, and PRR7 [80]. Long intron retaining CCA1/LHY transcripts provide examples of stress-responsive NMDinsensitive mRNAs [1,8,24]. An IR form of the mRNA of the master circadian regulator CIRCADIAN CLOCK

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Environmental cues, stresses

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Illustration of possible circuits involved in compensation of circadian oscillations through unproductive pre-mRNA splicing and NMD [24]. The key parameters — period, amplitude, and phase — define major properties of the circadian oscillator. Period, phase, and peak width of oscillating mRNAs must be sustained under environmental stress or during pathogen infection. Steady daily oscillations of the protein coding circadian mRNAs are achieved under environmental stress by a reversible shunting of the AS toward PTC+ transcripts. PTC+ mRNA can be degraded by the NMD machinery or escape NMD (NMD insensitive pool). Some incompletely spliced IR mRNA can be sequestered within the nucleus, to have their processing completed and be released at a later time. These mechanisms allow rapid adjustments in oscillations of productive mRNA and stabilize the circadian clock regulator under environmental stress at post-transcriptional level. www.sciencedirect.com

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ASSOCIATED 1 (CCA1) under normal physiological conditions mirrors oscillation profiles of its functional counterpart. However, broad diurnal temperature fluctuations alter not only relative levels but also timing of the peak levels of the IR isoform of CCA1. Filichkin et al. demonstrated that the IR isoform of CCA1 binds in vitro to splicing factor SR45 [24]. SR45 is distributed between nuclear speckles and nucleoplasm, and its localization is affected by stress [81]. Temporal phase delay of the IR isoform of CCA1 mRNA suggests that the SR45-bound CCA1 isoform can be sequestered and released ‘upon demand’ at the required times of day and/ or during thermal stress. How unproductive AS and NMD likely impacts the clock is outlined in Figure 3. The CCA1/LHY proteins could be possible candidates for the dominant-negative circuit of regulation at the protein level because the PTC in the retained long intron divides their N-terminal DNA-binding and C-terminal protein–protein interaction domains. However, the stability and functionality of this DNA-binding domain peptide remains to be demonstrated. A model that includes an additional circuit of temperature-driven, out-of-phase accumulation of intron retaining CCA1 nonsense transcripts has been proposed previously [24].

Initiative Competitive Grant # 2008-01077) to TCM and SAF, by the United States National Science Foundation (Plant Genome Research Program Grant # DBI-0605240 and # IOS-1025965) to TCM, and the United States National Institutes of Health (NIH R00 Award GM0971880) to MM.

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1. 

Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW et al.: Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 2010, 20:45-58 Available: http://www.ncbi. nlm.nih.gov/pubmed/19858364. One of the first comprehensive surveys of AS in plants using NGS approach. This study highlights unproductive stress-driven AS in plants. 2.

Marquez Y, Brown JWS, Simpson C, Barta A, Kalyna M: Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 2012, 22:1184-1195 http://dx.doi.org/10.1101/gr.134106.111.

3.

Rogers MF, Thomas J, Reddy AS, Ben-Hur A: SpliceGrapher: detecting patterns of alternative splicing from RNA-Seq data in the context of gene models and EST data. Genome Biol 2012, 13:R4 http://dx.doi.org/10.1186/gb-2012-13-1-r4.

4.

Reddy ASN, Rogers MF, Richardson DN, Hamilton M, Ben-Hur A: Deciphering the plant splicing code: experimental and computational approaches for predicting alternative splicing and splicing regulatory elements. Front Plant Sci 2012, 3:18 Available: http://www.pubmedcentral.nih.gov/articlerender. fcgi?artid=3355732&tool=pmcentrez&rendertype=abstract (accessed 24.07.12).

Concluding remarks Pre-mRNAs of the majority of Arabidopsis SR proteins are themselves extensively alternatively spliced [1,30,33], with approximately half of their isoforms escaping NMD [53]. At least 17% of alternatively spliced multi-exon, protein-coding Arabidopsis genes produce isoforms that are targeted by UPF-mediated NMD [55]. Recent findings suggest that UPF1 and UPF3 may also function in a translation-independent manner [55]. This raises the possibility that stable truncated polypeptides are involved in regulatory circuits at the protein level (Figure 3). Incompletely spliced pre-mRNAs may be temporarily sequestered within the nucleus, only to have their processing completed and be released at a later time. The expression levels of several non-circadian key regulatory genes, such as human SC35 [82] and Arabidopsis SR30 [1], are regulated via shunting of the splicing products toward PTC+ isoforms. Therefore, a model of reversible switches between productive and unproductive splicing may be applicable to non-oscillating systems. Identification of factors controlling spliceosomal responses to individual biotic and abiotic stresses will provide crucial insights into the mechanisms of stress-regulated unproductive AS. These recent findings suggest a complex network consisting of entirely posttranscriptional regulatory effects that have widespread impact on cellular adaptation to environmental stressors. In this network, multiple feedback loops link translation to RNA surveillance to RNA processing for both coding and non-coding transcripts.

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