Alternative
splicing
in
plants
–
coming
of
age
Naeem
H.
Syed
1,
Maria
Kalyna
2,
Yamile
Marquez
2,
Andrea
Barta
2and
John
W.S.
Brown
1,31
DivisionofPlantSciences,UniversityofDundeeattheJamesHuttonInstitute,Invergowrie,DundeeDD25DA,Scotland,UK 2
MaxF.PerutzLaboratories,MedicalUniversityofVienna,DrBohr-Gasse9/3,A-1030Vienna,Austria 3CellandMolecularSciences,TheJamesHuttonInstitute,Invergowrie,DundeeDD25DA,Scotland,UK
More than60% of intron-containinggenesundergo al-ternative splicing (AS) in plants. This number will in-crease when AS in different tissues, developmental stages,and environmentalconditions areexplored. Al-thoughthefunctionalimpactofASonprotein complex-ity is still understudied in plants, recent examples demonstrateitsimportanceinregulatingplant process-es.ASalsoregulatestranscriptlevelsandthelinkwith nonsense-mediateddecayandgenerationof unproduc-tive mRNAsillustratetheneed forbothtranscriptional andASdataingeneexpression analyses.AShas influ-encedtheevolutionofthecomplexnetworksof regula-tionofgeneexpressionandvariationinAScontributed toadaptationofplantstotheirenvironmentand there-forewillimpactstrategiesforimprovingplantandcrop phenotypes.
Frequencyandconsequencesof alternativesplicing (AS)
AS(seeGlossary)producesmultiplemRNAsfromthesame
genethroughvariableselectionofsplicesitesduring pre-mRNAsplicing.Itplaysakeyregulatoryrolein modulat-inggeneexpressionduringdevelopmentandinresponseto environmentalsignals[1–4].RegulationofASindifferent cell types andunder different conditions dependson se-quence elements in pre-mRNAs and the interactions of RNA-binding proteins whichvary intheir concentration and activity. The phenotype of a cell is determined by transcriptional,post-transcriptional,and post-translation-al networks,whichincludeasakeycomponentthe regu-latedASofthousandsofgenes.Inhumans,where>95%of genesarealternativelyspliced,extensiveproteindiversity is largelya resultofAS [5].Nextgeneration sequencing hasrevolutionizedresearchintoASandglobalmappingof human splicing regulatory proteins totheir target RNA sequences has ledto the development ofa splicing code that will allowpredictionoftissue-dependentAS [6].AS not only contributes to proteome diversity but also can generatetruncatedproteinsthatarepotentiallyregulatory or detrimental to the cell. It also plays a role in gene expressionbyregulatingtranscriptlevelsthrough produc-tion of isoforms which are degraded by the nonsense-mediateddecay(NMD)pathway.
Inplants,computationalanalysesofASeventsbasedon expressedsequencetagsandmorerecently high-through-puttranscriptomesequencinghaveexaminedthe frequen-cyofoccurrenceofASindifferentspecies[42%and33%of intron-containinggenesinArabidopsis(Arabidopsis thali-ana)andrice(Oryzasativa),respectively]andofdifferent typesofASevents[7–10].InArabidopsis,thefrequencyof occurrence hasrisensignificantly overthe past10years
(Figure1). Recently, an extensive RNA-seqanalysis has
significantly increased the observed frequency of AS in Arabidopsistomorethan 61%ofintron-containinggenes showing AS. This estimate of 61% of AS is based on analysisofplantsgrownundernormalgrowthconditions
[11]anditislikelythatthislevelwillincreasefurtheras different tissues at various developmental stages and growthconditionsareanalyzed[11].Ofthemostcommon types of AS (Figure 2b), intron retention (IR) has been showntobethemostfrequentASeventinplants[7–10]. However,someIReventswererecentlyshowntobemore likelytorepresentpartiallysplicedtranscriptsduetotheir
Glossary
Alternative50and30splicesites:useofalternativesplicesitesateitherendof theintron(intheintronorexonsequence)addsorremovessequences. Alternativesplicing:precursormRNAs(pre-mRNAs)arespliceddifferentlyto generatedifferentmRNAisoforms.
Exonskipping/inclusion:anexoncanberemovedinasinglesplicingeventor includedbytwosplicingevents.Suchexonsarecalledalternativeexonsor cassetteexons.
Heterogeneous nuclear ribonucleoproteins (hnRNP): RNA-bindingproteins whichbindRNAinthecell.
Intronretention(IR):oneormoreintronsis/arenotremovedfromapre-mRNA. It is oftendifficultto determine whetherintron retentionisdue to DNA contaminationorpartialsplicingofpre-mRNAsatthetimeofRNAextraction orareactivelyretained.
Micro-protein(miP):micro-proteinsareusuallygeneratedbytranslationofa transcript containing a premature termination codon, lack one or more functionaldomains,andhavefewerthan100aminoacids[82].
Nonsense-mediateddecay(NMD):acellularqualitycontrolmechanismthat recognizesmRNAtranscriptscontainingPTCsandtargetsthemfordegradation. Polypyrimidinetract-bindingprotein(PTB):anhnRNPproteinwhichbinds pyrimidine-richsequencesinRNAtoregulatealternativesplicingandother mRNAbiogenesisprocesses.
PrecursormessengerRNA(pre-mRNA):theprimarytranscriptofagenewhich isprocessedtomRNA.
Premature termination codon (PTC):a translational stop codonfound in transcriptsupstreamoftheauthenticstopcodon;PTCscanbegeneratedby mutationsinDNA,errorsintranscriptionorsplicing,oralternativesplicing. Serine/arginine-rich(SR)proteins:constitutiveandalternativesplicingfactors containingRNA-bindingmotifsandaRS-richdomain.
Small-interferingpeptides(siPEPs):shortpeptideswhichinterferewithcellular processes.
Correspondingauthor:Brown,J.W.S. ([email protected]), ([email protected]).
lowabundance[11].Inaddition,inthegenome-wide analy-sisabove,IRwasstillthemostfrequentASevent(40%)but itonlyoccurredinassembledAStranscriptsof23%ofthe genesprovidingamorereasonableestimateoftheimpactof IRtoASplants(Figure2c)[11].Moreimportantly,51%of intron-containing genes utilize alternative 50 or 30 splice sitesorexonskippingeventswhichcanaffecttheprotein
codingsequenceorgenerateunproductivemRNAstoaffect transcriptlevels[11].ThewidespreadoccurrenceofASand therangeoffunctionalgenegroupswhichitaffectssupports anessentialroleforASinplantdevelopment,physiology, metabolism,andresponsestoenvironmentalconditionsand pathogens, all ofwhich have important consequences on plant/cropphenotypes[7,12–18]. 0 10 20 30 40 50 60 70 2003 2004 2006 2010 2012 1.2% 11.6% 30% 42% 61% Improved technologies, various conditions, developmental
stages and tissues
% of genes with AS Year 30% 42% 61% gies, Improved technologie men tal
various conditions, developme stages and tis
sues
TRENDS in Plant Science
Figure1.Increasingfrequencyofoccurrenceofalternativesplicing(AS)inArabidopsiswithtime.In2003,astudyusingEST(expressedsequencetag)librariesestimated thatonly1.2%ofthegenesinArabidopsisundergoAS[101].Subsequently,greatercoverageofESTsandcDNAslibrariesallowedthediscoveryofmanymoreASevents (2004–2006,[7,102–104]).Theadventofhigh-throughputtechnologies[9,11]hasresultedinsignificantincreasesinthefrequencyofAS(almost60-foldoverthepast10 years). Types of AS events (a) Intron-containing genes AS transcripts AS events +IR 23.6% –IR 74.6% IR 40% Other AS 60% +IR 9.9% –IR 51.3% AS 61% (c) (b) IR Alt 5′ ss Alt 3′ ss ES ESE ESS ISS 5′ GU AG A (Y)n 3′ ISE + – + – UA-rich
TRENDS in Plant Science
Figure2.Maintypesofalternativesplicing(AS)eventsandfrequencyinArabidopsis.(a)Splicingofpre-mRNAisdirectedbyciselementswhichincludesplicesites,branch point,andpolypyrimidinetractsequences.Selectionofalternativesplicesitesisaffectedbytrans-actingfactorsbindingtoauxiliaryexonicandintronicciselements, termedsplicingenhancersandsilencers.(b)TypesofASevents.(c)FrequencyofoccurrenceofintronretentioninArabidopsis.IntronretentionisthemostfrequentAS eventinArabidopsis(40%)butitscontributiontotranscriptdiversityismuchlower[11].Ofthe61%ofArabidopsisintron-containinggeneswithAS,51%produceAS transcriptswhichdonotinvolveintronretention(–IR).Amongalternativelysplicedtranscripts,23.6%containoneormoreretainedintrons(+IR),whereastherest(74.6%) areproducedbyotherASevents.Coloredboxes,exons;lines,introns;GU,50splicesitewhichincludeshighlyconservedGUdinucleotide;AG,30splicesitewhichincludes highlyconservedAGdinucleotide;A,branchpointadenosine;(Y)n,polypyrimidinetract;ovals,positiveandnegativesplicingregulators;carets,splicingevents;thickgray line,unspliced(retained)intron.Abbreviations:ESE,exonicsplicingenhancers;ESS,exonicsplicingsilencers;ISE,intronicsplicingenhancers;ISS,intronicsplicing silencers;Alt30ss,alternative30splicesites;Alt50ss,alternative50splicesites;ES,exonskipping;IR,intronretention.
Regulation ofalternativesplicing
Assembly of the spliceosome during intron removaland ligationofexonsisdirectedbysequencefeaturesofthe pre-mRNA.Thecissequencesinthepre-mRNAincludesplice sites, branch point, polypyrimidine tract, enhancer, and suppressor sequences (Figure 2a). In addition, it is well documentedinplantsthatUA-richnessofintrons contrib-utestotheirrecognitionandisessentialforefficient splic-ing.Thesplicesitessituatedatthetransitionbetween UA-richintronsandGC-richexonsarepreferentiallyselected forsplicing(forreviewsee[19]).Thecompositionalbiasfor UA-richnesshasbeenalwaysconsideredasa distinguish-ingfeatureofplantintrons;however,recentlyithasbeen shownalsoforanimalsthatexonshavehigherGCcontent than introns [20,21]. Regulation of AS depends on cis signalsandtheirrecognitionbytrans-actingsplicing fac-tors.Serine/arginine-rich(SR)andheterogeneousnuclear RNP (hnRNP) proteins function as constitutive and AS splicing factorscontrolling splicesite choiceina concen-tration-dependent manner [22]. SR proteins are highly conserved in metazoa andplants and, typically, contain oneortwoRNArecognitionmotifs(RRMs)anda C-termi-naldomain(CTD)richinserineandarginineresidues(RS domain). Interestingly, plants possess plant-specific SR proteinsandingeneraltheyhavealmostadoublenumber ofSRproteinsofthatinnonphotosyntheticorganisms[23]. Many of them have different spatiotemporal expression patterns, implicating diversetargetspecificities and bio-logicalfunctions[24].hnRNPproteins,bycontrast, consti-tuteastructurallydiversegroupofRNA-bindingproteins associatedwithnascentpre-mRNA moleculesand,in ad-ditiontotheirroleinsplicing,areinvolvedinavarietyof molecular processes [25]. SR and hnRNP proteins bind splicingsignalsandintronicandexonicenhancer/silencer sequencesandthroughmulticomponentinteractionswith other splicing factors (including cell- and tissue-specific factors) determine splice site selection and where the spliceosome assembles. In general, SR proteins promote splicing andthehnRNPproteinsinhibitsplicesite selec-tion.However,bothSRsandhnRNPscanalsohave oppo-site functionswith, forexample,SRSF10(also known as SRp38)[26]beinganegativeregulatorandpolypyrimidine tract-bindingprotein(PTB;alsoknownashnRNPI)beinga positiveregulator[27].
Variationinabundanceand activity ofsplicingfactors determines theAS profilesof target genes and therefore theirdifferentialexpressionunderdifferentgrowth condi-tionsandduringdevelopment[28–31].Importantly, differ-entgrowthconditionsmodulateASofSRandhnRNPgenes causing dynamic changes in the splicing factor profile, further impacting expression of target genes (Figure 3). Forexample,ASofmanyArabidopsisSRgenesisaffected bytemperature,light,salt,hormones,etc.[30–32].In addi-tion,theactivityorlocalizationofSRproteinscanbe affect-edbyphosphorylation[4,33,34]andproteinkinaseswhich phosphorylateplantSRproteinshavebeenidentified[35– 37].Notsurprisinglytherefore,SRproteinoverexpression and knockoutlines showa varietyofdevelopmental and growthphenotypes,demonstratingtheimportanceofthese proteins to normal growth and development and broad effects on gene expression [28]. The best-studied plant
hnRNP proteinstodate are the Arabidopsisorthologs of theanimalnegativesplicingregulator,PTB[38],and the glycine-richRNA-bindingproteins,GRP7andGRP8, com-ponentsofaslaveoscillatorcoupledtothecircadianclock
[39–41].TheArabidopsisPTBsauto-andcrossregulatethe
ASwithinthefamily[38].GRP7andGRP8also autoregu-late their own AS and crossregulate each other’s AS to generate unproductive mRNAs which are targeted by NMDtoreducemRNAandproteinlevels[39,41].Finally, thenuclearcap-bindingcomplex(theCBC)consistsoftwo subunits,AtCBP20andAtCBP80,andAtCBP20containsa canonical RNA-bindingdomain (RBD).Mutation ofthese subunitsshowedtheCBCtobeinvolvedinASofatleast someArabidopsisgenesandtopreferentiallyinfluenceASof thefirstintron,particularlyatthe50splicesite[42].
Althoughtheabundanceandactivityofsplicingfactors determinestheASprofilesofdownstreamgenes,veryfew
Environment development cell/tissue type Direct activation/ deactivation of SFs Transcription/AS of SF genes Altered SF profile/ activity Phenotype response Altered RNome/ proteome AS of target genes
TRENDS in Plant Science
Figure3.Dynamicregulationofexpressionbyalternativesplicing.Developmental orenvironmentalcuesactivatesignalingpathwayswhichcandirectlymodulate splicing factor activity by post-translational modifications, relocalization, etc. Signaling also directs changes in transcription of splicing factor genesand alternative splicingofthesegeneschangestheabundance, composition,and activity of the splicing factor population. Expression of other target genes including transcription factors is also modulated by alternative splicing respondingtothedynamicchangesinsplicingfactorprofiles.Changesinthe proteome feedback to transcription and alternative splicing(and other post-transcriptionalmechanisms)ultimatelygeneratingthecellularand organismal phenotypeandresponse.Boxesofcoloredbars,abundanceand/orcompositionof SFs.Abbreviations:AS,alternativesplicing;SF,splicingfactor.
examples of the biological relevance of AS-derived pro-tein isoforms of splicing factors have been published. Functionaldiversity ofAS isoforms waselegantly dem-onstrated when two isoforms of SR45 (which differ by onlyeightamino acidsandinclude putative phosphory-lation sites), differentially complemented petal or root developmental phenotypes in the sr45 mutant [43]. Hence, isoforms with very similar sequences can have substantially different morphological outcomes which willreflectahostofgeneexpressionchangesindifferent organsofthe plant.
Epigeneticcontrolofalternativesplicing
An extensive body of evidence from human and yeast showsthat splicingisoftencoupledtotranscription[44– 46].TheCTDofRNApolymeraseIIservesasalandingpad forrecruitment ofproteins involvedin capping,splicing, polyadenylation,andexport[47–52].Therateof transcrip-tionelongationbyRNApolymeraseIImayaffectsplicesite choiceandtherebyASoutcomes[45,46].Aslowerrateof transcription tends to favor inclusion of weak upstream exonsbeforethesplicingcomplexiscommittedtosplicing ofastrongerdownstream exon[45,46].Importantly, effi-ciencyofthesplicingprocessmayalsoinfluencetherateof transcription elongation, where, for example, transient pausing of RNAP II at the 30 splice site of an intron coincides with the appearance of spliced product [53]. Therateoftranscriptelongationmaydependonthe chro-matinstate[50].Forexample,nucleosomeoccupancy var-ies along a gene with GC-rich exons being relatively nucleosome-rich compared with GC poor introns
[20,21,52,54] and transcription through nucleosome-rich
regions with compact chromatin tendstobe slower [50]. Furthermore,nucleosomeoccupancyisalsolowerin alter-natively spliced exons compared with constitutively splicedexons[20].Therelationsbetweenchromatinstate, nucleosomeoccupancy,RNAPIIelongationrates,splicing efficiency,andASoutcomesarekeytounderstandingAS regulation.Indeed,adirectlinkbetweenhistone modifica-tionsandASwasdemonstratedrecently[55].Thesplicing of two PTB-dependent mutually exclusive exons in the humanfibroblastgrowthfactor receptor 2(FGFR2)gene depended on histone modifications H3K36me3 and H3K4me1 acting in one direction, and H3K27me3, H3K4me3,andH3K9me1actingintheoppositedirection. Thechangesinchromatinstatearereadbythe chromatin-bindingadapterprotein MRG15which thenrecruitsthe splicingfactorPTBtothepre-mRNA andaffectssplicing outcomes[45,46,55].Inplants,thedirectlinkbetweenAS andeither chromatin state or RNAP IIelongation rates (transcription)has notyet beendemonstrated.However, clearly the impact of environmental cues and signaling pathways on chromatin and transcription to generate different AS variants has the potential to further our understandinghowplantsrespondtotheirever-changing environment.
Alternativesplicingaffectsproteincomplexityand transcriptlevelsandstability
Thedifferent formsofAS (Figure 2b) and, inparticular, alternative50and30splicesiteselectionandexonskipping
oftenleadtochangesinproteinsequencesfromthe inclu-sionorremovalofafewaminoacids(seeaboveforSR45
[43])tolargeregionsofproteinsaffectingproteindomains, orgeneratingchangesinN-terminalorC-terminalregions
[56,57].Differentprotein isoforms havethe potentialfor
differential functions as highlighted by several recent studies.Forexample,cold-inducedsweeteningisaserious problem inpotatoeswherestarchisconverted toglucose and fructose by vacuolar acid invertase: lines showing resistancetocold-inducedsweeteninghavehigher expres-sion of two splice variants (INH2a and INH2b) of the invertaseinhibitorgene(INH2)[58].Theflavin-dependent monooxygenasegene,YUCCA4,involvedinauxin biosyn-thesis, undergoes tissue-specific AStogenerate isoforms with different intracellular localization. One isoform is expressed in all tissues and is distributed throughout thecytosol,whereas asecondisrestrictedtoflowersand is attached to the endoplasmic reticulum [59]. In silico analysis of MADS-box MIKC-type (MADS, Intervening, Keratin-likeandC-terminaldomain)transcriptionfactors in Arabidopsis predicted protein isoforms which affect dimerization properties or higher order protein complex formation[57].ThepotentialforAStoinfluencefunction wasshownbythedifferentialeffectsonfloweringtimeand floraldevelopmentofoverexpressionofisoformsofSHORT VEGETATIVE PHASE, consistent with their different protein–proteininteractions[57].Thissystematicanalysis of a large gene family illustrates the potential of AS to affect key protein domains and function as well as the impactofASintheevolutionofgenefamiliesandprotein interactionnetworks.
AScanregulatemRNAlevelsthroughtheproductionof AS isoforms containing premature termination codons (PTCs) which are targeted for degradation by NMD
[60,61]. Plants possess orthologs of the key eukaryotic
NMDproteins,UPF1,UPF2,UPF3,andSMG-7(but not SMG-1,SMG-5,orSMG-6)andthesehavebeenshownto beinvolvedindegradingmRNAswithPTCs[62–68].Rules forNMDinplantshavebeenestablishedmainlyby study-ingmutationsinasmallnumberofmodeltranscripts.The mechanisms of recognitionof NMD substrates in plants appear to be fundamentally similar to those in other eukaryotes relyingonthe distance from aPTC tothe 30 end ofthe transcript (long30UTR)or downstream splice junctions(splicing-dependent)[18,64,69–71].Recently,by analyzing a large population of endogenous Arabidopsis transcripts,coupledAS,andNMDhasbeenshowntobea widespread mechanism for regulating gene expression with 11–18% of alternatively spliced transcripts being turned over by NMD [18]. This study also showed that transcripts containing PTCs which are NMD substrates areoftenreadilydetectableandcancontribute significant-ly tosteady-statetranscript levels of genes.In addition, somePTC-containingtranscriptswerenotturnedoverby NMD.Forexample,sometranscriptscontainingretained introns or parts of introns were unaffected in NMD mutants,suggestingthat notallNMDtriggeringsignals ortranscriptarrangementsareunderstood[18].Thus,the generation of unproductive AS transcripts can influence thelevelsoffunctionalmRNAs(full-lengthproteincoding), ashasbeenobservedintheregulationofhumanSRand
hnRNPproteinsthroughASandultraconservedelements
[72–74].Similarlyinplants,SRandPTBgenesare
regu-latedbyAS[28,38,75]whichgivesrisetoPTC-containing transcripts, suggesting a regulatory function via unpro-ductive mRNAs.
Alternativesplicingintheplantcircadianclock
Regulation of expressionby AS generating unproductive transcriptshasrecentlybeendemonstratedforcore circa-dian clock genes in Arabidopsis. Circadian clocks have approximately 24-hrhythms andalloworganisms to an-ticipatetheday–nightcycle andcoordinatetheirgenetic, biochemical, and physiological responses [76–78]. Core clock gene expression is regulated at multiple different levels: transcription, protein degradation, and modifica-tionwithASbeinganemergingthemeinregulationofthe clock[77,78].Untilrecently,examplesofASinclockgenes andtheirfunctionalsignificancewere rare.Forexample, anIReventinCCA1wasconservedinatleastfourplant speciesandlevelsofIR-containingtranscriptsincreasedin high light and decreased in the cold [9]. In addition, a
mutantinPROTEINARGININEMETHYL
TRANSFER-ASE 5 (PRMT5) showed a longer circadian period and dramatic changes inthe levels ofunproductive AS tran-scripts of PRR9 [79,80]. Recently, extensive AS in the majorityofthecoreclockgenesinArabidopsiswas identi-fied and dynamic changes in AS profiles for many AS events wereobservedinresponsetochangesin tempera-tureandparticularlytolowertemperatures[81].ASevents were eitherinducedbylow temperaturesor increasedin abundance to10–50% ofthe transcripts; the majority of theseeventswerenonproductiveresultinginareductionof functional mRNAs potentially impacting protein levels
[81]. Furthermore, the partially redundant gene pairs, LHYandCCA1,andPRR7andPRR9behaveddifferently with respect to AS, implying functional differences be-tween theserelatedgenes.Thus,temperature-associated AS modulatingthe balancebetween productiveand non-productive mRNA isoforms is an additional mechanism involvedintheoperationandcontroloftheplantcircadian clock.
Alternativesplicingandregulationbysmallinterfering peptides/micro-proteins
The fate of alternatively spliced transcripts containing PTCsisexpectedtobedegradationbytheNMDpathway but some PTC-containing transcripts are stable and ap-pear toavoid the NMD machinery [18]. PTC-containing transcripts also have the potentialto be translatedinto truncatedproteins orpeptides.Inplants, intron-contain-ing mRNA transcripts were found associated with poly-somes and recently ribosome profiling in mouse has identified novel upstream open reading frames (ORFs) and ORFs in long noncoding RNAs [82,83]. In animal and plant systems, small interfering peptides (siPEPs) or micro-proteins(miPs) named after theiranalogy with siRNAs and miRNAs have been described[84,85]. Such peptidescanhavealteredfunctionalitybyonlycontaining particular domains (e.g., DNA binding, transcriptional activators) and can act as both positive and negative regulators andaffect regulatoryfeedback loops [86].For
example,inanimals,anASisoformgeneratesamiPofthe ETS1 transcription factor which regulates growth and development responses, lacks the transactivation domains, and interacts physically with ETS1 blocking ETS1-mediatedexpression oftargetgenesinadominant negativemanner[86–88].Similarly,miP ASprotein iso-formsoftheanimaltranscriptionfactorMEIS2also inter-actinadominantnegativemanner[89].Interestingly,one ofthesplicevariants(MEIS2E)isstructurallysimilartoa plant protein‘KNATM’,whichisamember oftheTALE homeodomain transcription regulators (controlling meri-stem formation, organ position andmorphogenesis, and someaspectsofreproductivephase)[86].KNATM, howev-er, lacks a homeodomain and by forming nonfunctional heterodimerswiththeBELLTALEproteinregulatesleaf pattern[90,91].Itisintriguingthataproteingeneratedvia AS in animals appears to exist as a miP equivalent in plants.
Genome-wideanalysisofASinArabidopsis suggested that 78% of alternative transcripts introduced in-frame PTCs[9],providingahugepotentialforproductionofmiPs. ExamplesofmiPsinplantswithfunctionalconsequences arerare butarecentstudyshowed that theArabidopsis transcriptionfactorgene,IDD14,producesasplicevariant (IDD14b)whichlackstheDNA-bindingdomainbut inter-actswiththefunctionalIDD14aisoformtoproduce hetero-dimers.The IDD14a/b heterodimerhas reduced binding affinityfor thepromoterof theQua-QuineStarch (QQS) gene which regulates starch accumulation by initiating starch degradation [84]. Starch accumulation is one re-sponseofplantstocoldandastheIDD14bsplicevariantis onlyexpressedundercoldconditions,starchdegradationis reduced providing an AS/miP-dependent strategy for maintaining starch reserves at low temperatures. Inter-estingly,thecoreclockproteinsCCA1andLHYcanboth homo-andheterodimerize[92,93],anddifferent combina-tions have different binding affinity to their target sequences[94].Itisinteresting tospeculatewhetherthe extensiveproductionofPTC-containingtranscriptsincore clockgenesbyAScouldaddafurtherlevelofregulationby miPs.Furthermore,siPEPs/miPsofferanothermechanism tomodifyorknock-downexpressionofendogenousgenes. Artificial siPEPs/miPs encoding dimerization domains couldbetransformedintoplantstoreducetheactivityof a targetgene. As they function at the protein level and depend on homo- or heterodimerization, this should improvespecificityandreduceoff-targetsilencing[85]. Alternativesplicingdiversityinecotypesandpolyploids Extensive ASoccurs underalteredgrowth orstress con-ditionsin plants.Similarly, extensive variation in AS is expected in diverse ecotypes adapted to very different climatestherebyachievingenvironmentalandphenotypic plasticity. To address such diversity, genomic and tran-scriptomicsequencingisbeingperformedon geographical-ly and phenotypically diverse accessions of Arabidopsis. Sequencingofthegenomesandtranscriptomesof18 Ara-bidopsis accessionsidentifiedextensive singlenucleotide polymorphism and indel variation among the genotypes
[95]. When compared with Col-0 (TAIR10) one-third of protein coding genes were disrupted/altered in at least
oneaccessionalthough re-annotationrestoredcoding po-tentialinmostcases.Sequencevariationsaffected trans-lationstartandstopsites,introducedPTCs,orchangedthe frame of the coding sequence, or potentially generated protein isoforms in different accessions. Two-thirds of 2572geneswithdisruptedsplicesiteswhencomparedto TAIR10hadnewsplicesitesandaquarterofthesesites wereclose tothe splicesitesin Col-0[95].Clearly, natu-rallyoccurringsequencevariationcandisruptsplicesites andRNA-bindingmotifsfor splicingfactors. Such muta-tionsimpactproteinexpressionandactivityandprovidea basisfor selectionfor adaptationofdifferent ecotypes to theirenvironments[7,8,10,57].
Extensiveduplicationor polyploidizationhasoccurred in the evolutionary history of many plant species [96]. Theoretically,sucheventscould generateimmediateand drasticchangesintheabundance,composition,and activi-tyofsplicingfactorswhichinturncouldaffectsplicesite choice among variable analogous splice site sequences. Furthermutation,geneloss, orchanges inexpressionor protein functionality provide the basis for selection and continuedevolutionofthespecies.Recently,ASpatterns havebeenstudiedamongnaturalandsyntheticpolyploids ofBrassicanapus. Interestingly,two independently syn-thesized lines showed parallel loss of AS events after polyploidy [97]. This is intriguing because it showsthat even in two independent events ofpolyploidy, usingthe samespecies,resultsinidenticalpatternofASloss, point-ingtowardsanon-randomresponseoftheso-called ‘geno-micshock’aftertwogenomesphysicallyinteractwitheach other. The same study also showed that 26–30% of the duplicatedgenesshowchangesinAS,comparedwiththe parents,withsomeshowingorganspecificityorresponseto abiotic stress [97]. It is likely that AS has played an importantroleintheevolutionandadaptationof cultivat-edcropstodifferentenvironmentalconditionsandniches, becausemanycropspeciesare polyploidsandthiswhole areawillbeoneofgreatinterestinthefuture,particularly withthepowerofhigh-throughputsequencing.
Evolutionaryaspectsof alternativesplicingfactor diversity
TheArabidopsisgenomeencodes18SRproteinswhich is nearlydoublethenumberfoundinhumans.Atleast12ofthe 18SRgenesarelocatedinduplicatedgenomicregionsand thecurrentdataindicatethatmostofthemhavedifferent spatiotemporalexpressionpatterns,suggestingfunctional diversification[24].Indifferentplantlineages,thenumber ofparalogousSRgenesishighlyvariable.Forexample,the plant-specificRSsubfamilyofSRproteinsisencodedbyfour genesinArabidopsis,twoinrice,atleastfiveinPinustaeda andbyonegenebothinPhyscomitrellapatensand Chlamy-domonas reinhardtii[98].Differential or common, redun-dantfunctionsofSRparalogsindifferentspeciesremainto bedetermined.However,itisinterestingthatseveralSR paralogsandorthologsareregulatedbyASeventsconserved fromunicellulargreenalgae tolandplants.These events occurin theanalogous long intronssituated inthe RRM codingregions[98,99]and,moreover,theyinvolveunusually highlyconservedsequencesaroundalternativesplicesites
[98], suggesting an important biological function of such
regulation. Arecent systematic surveyofSR genes in27 eukaryoticgenomesshowedthatfloweringplantson aver-age possessnearly doublethe number ofSR genes than nonphotosyntheticspecies[23].Moreover,mostoftheplant SRgenesareunderpurifyingselection,ensuringthat para-logousgeneswhichoriginatedduetotheduplicationevents maintaintheirstructureandfunction,whereasredundancy isreducedviadiversificationofgeneexpression[23]. Futurechallengesinalternativesplicingresearchin plants
ASresearchinplantshasmadesubstantialprogressinthe past4–5years.Theever-increasingnumberofplantgenes withASandtheprocessesinwhichtheyareinvolvedpoint tothe importanceofunderstandingthe mechanisms and regulation of ASand the functions ofAS.The functional impactofASisoneofthemostimportantquestions–thisis largelyduetotherelativelysmallnumberofexamplesofAS forwhichdifferentialfunctionshavebeendemonstratedfor differentproteinisoforms.However,wedrawaparallelwith researchtodiscovertheextentofASinplants.Around10 yearsagothe firstestimatewasonly 1.2%ofplantgenes showing AS! With massively improved technologies this numberhasnowgrowntomorethan61%(Figure1). Simi-larly,theincreasingnumberofplantgenomesequencesand thegenerationofvasttranscriptomedatawillallow compu-tational analyses to identify conservation of AS events acrossspecies and tissue-,developmental-stageand envi-ronment-specific regulation of AS providing evidence of functionality. The number of functional examples of AS, whetheratthemRNAtranscriptstabilitylevelorprotein function,continuestogrowandinturnisstimulatingwider interest in AS in theplant community. High-throughput sequencing will also address dynamic changes in AS in developmentandunderdifferentenvironmentalconditions andstresses,andhowvariationinASpatternsindifferent ecotypesandpolyploidscontributestoplasticityand adap-tationofplantspecies.Furthermore,weneedtounderstand howsignalingpathwaysaffectsplicingfactoractivity direct-ly or viachromatin modification and how transcriptional andASnetworksinteract[100].ASisamajormechanismby which plants modulateand fine-tune expression oftheir genes.Thenext5yearswillseeanexplosionofknowledgeof the functional significance of AS and understanding its contributiontothecomplexityofgeneexpressionwilloffer newopportunitiesinapproachestomodifyingplantfunction forimprovedphenotypes.
Acknowledgments
ThisworkwassupportedbytheBiotechnologyandBiologicalSciences Research Council(BBSRC)[BB/G024979/1(ERA-NETPlant Genomics (PASAS)];theScottishGovernmentRuralandEnvironmentScienceand AnalyticalServicesDivision(RESAS);theAustrianScienceFund(FWF) [SFB1710,1711;DKW1207;ERA-NETPlantGenomics(PASAS)I254; SFB RNAreg F43-P10]; the Austria Genomic Program (GENAU III) [ncRNAs]; and the EU FP6 Program Network of Excellence on AlternativeSplicing(EURASNET)[LSHG-CT-2005-518238].
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