EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 1 Integrative analysis revealed the molecular mechanism underlying RBM10-mediatedsplicingregulation YongboWang,AndreasGogol-Döring,Hao Hu,Sebastian Fröhler,YunxiaMa,Marvin Jens,Jonas Maaskola,YasuhiroMurakawa,ClaudiaQuedenau,MarkusLandthaler,VeraKalscheuer,Dagmar Wieczorek,YangWang,YuhuiHuandWeiChen Correspondingauthor:WeiChen, MaxDelbrück CenterforMolecularMedicine Review timeline: Submissiondate: 20 February 2013 EditorialDecision: 26 March 2013 Revisionreceived: 07 June 2013 EditorialDecision: 06 July 2013 Accepted: 11 July 2013 TransactionReport: (Note:With the exception ofthe correction oftypographicalor spelling errors thatcould be a source ofambiguity, letters and reports are not edited. The originalformatting oflettersand referee reportsmaynotbe reflected in this compilation.) Editor:RobertoBuccione 1stEditorialDecision 26 March 2013 ThankyouforthesubmissionofyourmanuscripttoEMBO MolecularMedicine.Wehavenow heard back from thethreeReviewers whom we asked to evaluate your manuscript. YouwillseethattwoReviewersaremoresupportiveofyouworkwhileoneisquitenegative. Nevertheless,allthreeraisesignificantissuesthatquestiontheconclusivenessoftheresultsthus preventing usfrom considering publication atthistime.Iwillnotdwellinto much detail,asthe evaluationsare detailed and self-explanatory.Iwould like,however,to highlighta few main points. Reviewer1ismainlyconcernedaboutthemedicalrelevanceofthe observations.Specifically,s/he pointsto theincompletecharacterisation ofthepatientwhosecellswereused.In addition,Reviewer 1 would likeyou to considerthepotentialconsequencesofalteration ofsplicing,dueto RBM10,of othergenesinvolved in congenital anomalies. S/he also notes the imprecise citation of previously reported information and other issues thatrequire your action. Reviewer2isespeciallyconcernedwithoveralldatasignificance,completenessandqualityand providesadetailed explanation and list of required remedies; I will just focus on the main points. FirstlyandsimilarlytoReviewer1,s/hefeelsthatthemedicalangleofthestudy(whichforEMBO MolecularMedicineisofhighimportance)isnotsufficientlydiscussed and integrated.In this respect,I agree with Reviewer 1's assessmentthatrelevantmedicalexpertise mightbe usefulin revising the manuscript.Reviewer 2 also notes thatfor the PAR-CLIPpart,themotifanalysis requires more explanation and further analysis.Also,s/he isofthe opinion thatthe resultsofthe mini-geneexperimentshavebeen overestimated/overstated;Remediesaresuggested in thisrespect too. Reviewer 2 also points to flawed TARP syndrome analysis, including the interpretation of the consequencesofthe patientdeletion.ThisRevieweralso listsmany othercriticalpointsthatrequire youraction. EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 2 Reviewer3alsopointstotechnicalflawsandissuesofoveralldatasignificanceandcompleteness. Themanycriticalissuesmentionedincludethequality ofcontrols.Again,although Iwilljustfocus on themain points,allitemsrequireyourattention and action.Similarly to Reviewer2,Reviewer3 notesthatthePAR-CLIPpartrequiresextensiveclarificationincludingexperimentationwhere necessary and isalso concerned aboutthedataon theRBM10 binding siteclusters.Anotheritem of strong concern,again in accord with Reviewer2,is the outcome ofthe mini-geneexperiments, whichappeartohavemultiplecriticalities.Finally,inarecurrenttheme,Reviewer3 isnotsatisfied withtheconnectiontoTARPsyndrome.S/healsolistsotherexperimentalshortcomingsand suggests a numberofimprovements thatrequire action to increase the overallquality ofdata and presentation. Consideredallthe above,while publication ofthe papercannotbe considered atthisstage,we wouldbepreparedtoconsiderasubstantiallyrevisedsubmission,withtheunderstandingthatthe Reviewers'concernsmustbefullyaddressedwithadditionalexperimental datawhereappropriate and thatacceptance ofthe manuscriptwillentaila second round ofreview. Sincetherequiredrevisioninthiscaseappearstorequireasignificantamountoftime,additional workandexperimentationandmightbetechnicallychallenging, I would therefore understand if you chose to ratherseek publication elsewhere atthisstage.Should you do so,we would welcome a messagetothiseffect. PleasenotethatitisEMBO MolecularMedicinepolicytoallow asingleroundofrevisiononly and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next,finalversion of the manuscript. Asyouknow,EMBO MolecularMedicinehasa"scoopingprotection"policy,wherebysimilar findings thatare published by others during review orrevision are nota criterion forrejection. However,Idoaskyoutogetintouchwithusafterthreemonthsifyouhavenotcompletedyour revision,to update us on the status.Please also contact us as soon as possible if similar work is published elsewhere. ***** Reviewer'scomments***** Referee#1(CommentsonNovelty/ModelSystem): Theinvitrostudiesuseacombinationofnovelapproaches.Thestudiesperformedonpatient derived lymphoblastsare unique,few patientswith mutation in RBM10 have been identified. Referee#1(Remarks): ThisverywellwrittenandillustratedpaperdescribestheeffectofRBM10onsplicingofcassette exonsand identifiesa numberofgenesdifferentially affected by overexpression orknock down of RBM10.Thelaboratorystudiesarenovelandveryrelevant. A weaknessofthispaperisthelackofcompletemedicalcontext.Whilethereisabriefmentionof the relevance to cancer, and the cancer genes are highlighted in thesupplementary table,themore relevantprimary contextof embryologic developmentis notappropriately addressed.This major weaknessshouldbeaddressedbyaskingtheclinicians,whoidentifiedthepatientsonwhom this workisbased,tocontribute to the written manuscript. In the introduction you state the "100% pre- orpostnatallethality in affected males".Thisiswrong, aslong term survivalhasbeen reported {Gripp etal.,American JournalofMedicalGenetics2011}. Thedescriptionofthe patientfrom whom the cellline wasderived isinsufficient.Isthispatient alive ordeceased?Isthe cousin indicated on the pedigree alive ordeceased?Whatare the typical malformationsseeninTARP,andwhatmajormalformationswerepresentinthepatientwhose cells you used? Itis clear from the data presented here thatRBM10 affects splicing of numerous genes.Some of these genes listed in the supplementary table (CASK; TBX3; CREBBP; FANCA; POMT1 to name a few obvious examples)are known to be causally involved in congenitalanomalies.This should be discussed.Thegenesknown to beassociated with congenitalanomaliesshould behighlighted in the table (in addition to cancer- associated genes).Can any ofthese genes,by virtue oftheirabnormal splicing,resultin themalformationsseen in TARP syndrome? EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 3 WhileIrealizethatthisisnotthefocusofthisparticularreport,thisisthecontextinwhichthework is relevant to human development and medicine. A clinicians'input in this manuscript isnecessary. Minorissues: page8:"anti-correlation"- do you mean inversecorrelation? Theterm "mentalretardation"isnotusedinprofessionalpublicationsanylonger;use"intellectual disability"instead. DefineTARPatitsfirstuse.Whatdothelettersstand for? page14;middleparagraph:thesentence"Thissuggested thismotif..."doesnotmakesenseas written. "Acknowledgement" is misspelled. Referee#2(CommentsonNovelty/ModelSystem): Themodelsystemsusedwereappropriateforthebiochemical requirements of the study. The technical quality of the paper can be vastly improved by appropriate informatic controls (as pointed outin remarksbelow).Thenovelty ismedium asmechanistically nothing wasreally teased apart. Referee#2(Remarks): In the manuscriptentitled "Integrative analysis revealed the molecular mechanism underlying RBM10-mediatedsplicingregulation"Wangetal.perform PAR-CLIPtoidentifygenome-wide binding sitesfortheRBM10 protein,and perform RBM10 KD and overexpression experiments followed by RNA-seq analysis to identify RBM10 dependentsplicing changes.The authors then use the genomics data to try to characterize the mechanisms of RBM10 action based on correlative analysesbetween RBM10 binding and alternative isoform usage.Finally,the authors perform a limited analysis of an apparent NLS deletion RBM10 mutation associated with human TARP syndrome,finding thatthis patientsample shares the alternative splicing profile ofthe RBM10 knockdown. Althoughseeminglypromising,themanuscriptlacksaclearand compelling biologicalstory thatis supported with solid evidence,and seems to be missing many more detailed analyses to make the results more convincing.The CLIP and RNA-seq experiments willprovide datasets forfurther research on RBM10,and the proposed link between RBM10 and the U2 snRNP is interesting (although notvery wellexplored experimentally).However,the RBM10 binding mechanism remains unclear (as no motif is identified from the intronic binding sites, and the GAAGA motif identified from exonic binding sites apparently didn't validate in gel shift assays), and the effect of RBM10binding,eveninthelimitedminigeneassayspresented,isweak(~5% effectonexon inclusion). In addition, while the majority ofthe manuscriptfocuseson intronic RBM10 binding,the profilein Figure2aissignificantly shifted towardsexonicbinding (~40% exonicvs50% intronic) asopposed to the whole human genome (lessthan 1:5 exonic to intronic),suggesting thatthe exonic binding sitesaremorelikely to bebiologically relevant. Similarly,theTARP syndromesectionfeelsabittackedon;itisinteresting(thoughperhapsnot surprising)thata patientsample with an RBM10 NLS deletion would resemble (atthe splicing level) RBM10 knockdown, but it remains unclear to what degree this altered splicing pattern actually leadsto phenotypic effectsassociated with the disease.Asthe authorsdon'tdiscussthe degreeto which geneexpression isaltered in thesesamples,it's not clear whether altered gene expression oraltered splicing isthe majorcomponentofTARP syndrome in thisindividual. Asageneralcomment,thesupplementalfiguresneedtobehigherresolution(asthey'reimpossible to read as presented here). Moredetailed commentsarebelow: PAR-CLIPanalysis: • For the binding near exons (Fig 2b),there seems to be a controlline missing.A shuffled controlor even anotherprotein'sPAR-CLIPdatasetwouldmaketheresultsforRBM10moreconvincing. EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 4 • In Figure 1a,Iassume thatUTRsare included in the exon annotations- whatdoesthedistribution look like if you separate them from exons? Is there any UTR enrichment? • The motif analysis seems to have been done in a very specific way in order to acquire some sortof enrichment.Imissed any description ofthe algorithm used formotiffinding otherthan 'pentamer enrichment'- wastheGAAGA motifmostsignificantlyenriched,greatestfold-enriched,etc?A statisticaltestshould also be included forsignificance ofenrichmentin exonsand in the vicinity of both 5'and 3'splicesitesoftheintrons.Theauthorsalso do notdescribetheirdefinition of strong/medium/weak binding sites.I'm also confused by the apparentperiodicity ofpentamer frequencies in 2c - is there an explanation for why this might be observed? Or is this an artifact of some unspecified windowing ornormalization procedure? • Fig 2d seems unnecessary as a main figure or even a supplementalfigure,as itis a very weak result. • The discussion includesmention ofagel-shiftassay thatfailed to detectdirectbinding ofRBM10 to the GAAGA motif described in figure 2c. This result needs to be presented in the results section during thediscussion offigure2c,asithassignificantimplications forinterpretation of figure 2c.It also needsto be furtherdiscussed,asatthe end ofthe paperitisunclearwhetherthe authorsbelieve that this reflects an RBM10 motif or rather a motif of some unspecified additional regulator that RBM10associateswith. • The inference of U2 snRNA binding by PAR-CLIPreaddensityinFigure2eisinteresting- forthe second partoffigure 2e,itwould be helpfulto include the data (read counts /base),as it's unclear whetherthetwoindicatedpositionsaremostenriched oraretheonly positionsobserved to havethe T->C crosslinktransitions. RBM10OE/KD RNA-seq: • The authors go straightto altered splicing upon RBM10 KD and OE - are there significant alterationsatthe gene expression level?Irecognize thatthefocusofthispaperison alternative splicing regulation by RBM10,butthe scale to which RBM10 manipulation generally effects gene expression would be valuable (aswellasinformation on otherRNA binding proteinssignificantly altered in the RBM10 KD/OE experiments). • In SupplementalFig.2a,the western blotfor the RBM10 KD and controlsample needs to be shown togetheron the same western blotas done in Supp Fig 2b.As itis,itappears thatthe authors pasted togethertwo lanesfrom potentially different gels, which may or may not show an actual KD atthe protein level.Quantification (particularly ofthe knockdown western blot)would also be ideal, asitappearsthatGAPDH isalso lowerin the KD sample. • Itseems thatthe overlap of events changed in theOE and theKD is306,butwhatabouttheevents that do not overlap (not in both the OE and KD experiment)- is there anything interesting with those? Are these just false positives or were they just missed in one of the experiments? Can they be validated in both conditions? • The authors report17 events thatvalidated via qPCR - however,itisdifficultto determinefrom the data shown how representative these events are, as 11 of the 17 have >~2-fold changes in PSO whereasthemajorityofevents in figure 3a have deltaPSI<|0.2|.Additionally,detecting splicing changesby qPCR requiresa fairamountofnormalization calculations,butthese validationscan be donemoresimply with RT-PCR andrunningagarosegels.Canthesplicingchangesbedetected this wayorweretheyonlydetectablebyqPCR? • Figure 3c shows a track labeled "ctrl" - is this the control for the OE or the KD? (The authors list independent control datasets for both experiments - hopefully separatecontrolswereused in all splicing experimentsthroughoutthe paper.Thisshould be made explicitand corrected ifnot). • Splicing maps are made with the RBM10 OE data,whatdo they look like with the KD data - hopefully thesame. • Whatfraction of the events thatchange are associated with RBM10 binding sites?Conversely,the PAR-CLIPexperimentidentifiedmany(~88k)bindingsites- whatpercentofbindingsitesare associated with regulated targets? RBM10splicingmodels: • The minigene experiments seem like a nice idea,butI unfortunately Idon'tthink theresultsareas convincing asclaimed.In Figure 4,the authorsstate that"These data provide unequivocalsupportto ourhypothesisthatRBM10 binding ...would facilitatetheskipping ofcassetteexons",butthelack oftechnicalqPCR errorbarsaswellassome sortofstatisticaltestto show whetherthe observed changesare significantconflictwith such a strong statement.Additionally,the effecton splicing for three of the four mutations made in Fig 4b/c is extremely small (~5% or less), and the data shown EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 5 only reflectsthechangein exon exclusion between OE and control.Iwascuriousasto whetherthe mutationsperformedshowsignificantlyalteredPSOratesinthecontrol(orover-expression)by themselves, and to what theratesofexon inclusion fortheseeventstypically are(i.e.,ifthey were 90% excluded in thecontrolthen asmalleffectupon RBM10 OE would benotsurprising). Additionally,itwouldbeidealtoperform thedirectexperiment(forcedRBM10association with intronic loci, e.g. by MS2 tagging) to prove that RBM10 recruitment to an intron will alter splice site choice. • P-valuesaremissing forFig.5d.Thestrength ofsplicing sitesdistalto thecassetteexonsdoesnot seem significantly strongerthan that of those immediately flanking the exons, and in general the effectsize here seemsto be very slight. • Itlooks like fig 6b should be recreated by the authors,seems like itwas taken from another publication. TARPsyndromeanalysis: • Intriguingly, the splicing changes observed here correlated well with changes induced by RBM10 KD inHEK293(Fig.6c).WhataboutcomparedtotheOEexperiment? • Supplementalfigure 3 needs better quantification - it is clear from the figure that the mutated RBM10isnotsilenced,butitisdifficultto tellwhethertheexpression isactually unchanged from the results shown. Figure 6c also needs statistics - whatisthecorrelationbetweenthetwosamples? • Figure 6c-e are used to propose thatthispatientdeletion actsasan RBM10 deletion through lossof nuclearexpression.However,thedeletion includesnotonly theNLS butalso additionalsequences. Figure6ewouldbestrengthenedbymutatingeitheronlytheNLS,orattachinganormalNLS tothe mutatedRBM10toshow thatthemolecularphenotypesobserved in thispatientarecharacteristic mis-localization of RBM10 and not also loss of function through deletion of additional domains. Discussioncomments: • Overall,the discussion needs to be rewritten to putthe results of the paper in context.The idea the RBM10workswithotherRBPsneedstobepresentedsoonerthanthediscussion,otherwisethe results section makes no sense in this context- forexample,itis notuntilthe discussion thatthe authorsimply thatRBM10 doesnotseem to bind the identified motifsby itself.Similarly,the TARP section needs a betterdescription ofhow itfits with the results in the restofthe paper- if RBM10 loss of function causing TARP is previously known, is the novelty thevalidation oftheindividual deletion patientasactually losing nuclearlocalization?Orthatthelossofnuclearlocalization of RBM10resemblesRBM10knockdown? • The statement"Whole-mountinsituexpressionanalysisofthemurineRbm10hasshownthatthe genewasexpressed during embryonicdevelopmentin apattern consistentwith thehuman malformationsobservedinTARPsyndrome"ismissingareference. • The discussion refers to two experiments thatneed to be incorporated into the main text,as they are notmentioned anywhere in the manuscriptbefore the discussion - oneexperiment(an in vitro binding RBM10 experiment)thatisnotshown in any figuresand only mentioned in the supplementalmethods,and discussion ofthe effectofexonic RBM10 binding (figure7),which is also notpresented in the results. Minorcomments: -Breitlingetal2004citationinthemethodssectionismisformattedandnotincludedinreference list -typo in figure reference: "In agreement with our in vivo data, the skipping ofthecassetteexonswas enhanced upon RBM10 OE (Fig.54b)" -Figure5figurelegendismislabeledasfigure4 Referee#3(CommentsonNovelty/ModelSystem): Technicalquality Majorissues:EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 6 1.Filtering outpotentialcontaminants.Whilemostofthereads are derived from cross-linked RNA species (carry U to C transitions),from supplementary figure 1a itis clearthatthere is successful RNA pull-down from thecontrolsamplesthatwerenotcross-linked. Such result indicates that the washingstringency may nothavebeen sufficientto removespeciesofRNA retained non- specifically on the resin orbound through mediated interaction.Reads derived from such non- specifically interacting RNAs can clearly be identified in PAR-CLIPbytheabsenceofU toC transition in the sequence. Were those reads removed from the set that was used to define the RBM10bindingsite? 2.Binding siteclusters.Theauthorsneed to providemoreinformation on theclusterthatwere identified in the PAR-CLIPexperiment: (i) Whatisthe distribution ofthe clustersizes? (ii) How many reads are forming a typicalcluster and whatis the read countdistribution of the clusters?The relatively smalloverlap between the two experimentswould indicate thatmostofthe clusterare formedbyrelativelyfewreads. (iii) Within each cluster,are there preferred cross-linking sites? This would be a strong indication of the presence of high affinity binding site. 3.RBM10 binding sitesequence.Theauthorsidentify GAAGA asapentamerthatis enriched in the RBM10bindingsiteclustersthatarelocatedwithinexons.TheabsenceofUridineresiduefrom the binding siteisquitesurprising considering thatPAR-CLIPisreliantoncross-linking to U residues. Itraises the possibility thatthe approach used to identify thebinding sitesequenceisinadequate.In particularwhatwastherationalto analyzeseparately theexonicand intronicbinding sites?Do the authorsexpectRBM10 to bind to a differentsequence in the intronscompared to the exons?Was the GAAGA pentamer significantly enriched in the intronic binding sites? If not, than what is the evidence ofsequence specific binding ofRBM10 to RNA? AlternativelyitispossiblethatthesequencerecognizedbyRBM10doesnotcontainuridinesthat can be cross-linked to the protein. This would mean that PAR-CLIPisnotanadequateexperimental approach foridentifying RBM10 binding sitesand the standard CLIP approach,thatusesshort wavelengthUV lighttocrosslinktounmodifiedRNA shouldbeused instead. 4.Binding to U2.Thefinding thatRBM10 bindsto U2 snRNA isquiteintriguing.How many reads weremappedtoU2comparedtothereadsmappedinproteincodinggenes.Consideringthehigh abundance ofthe snRNP RNAs,isthere enrichmentofRBM10cross-links to U2 that is statistically significant? 5.Correlation between RBM10 binding and effecton splicing.Onestrong aspectofthepresented workistheavailabilityofbothRNA binding(PAR-CLIP)andexoninclusiondata(RNA-seq). Surprisinglythe authors do not provide information as to what fraction of the RBM10 regulated exonscontain RBM10 binding sites.Good correlation between RBM10 binding and exon inclusion/skipping would strongly argue that the effect on splicing is specific to RBM10 and isnota secondary effect. 6.RNA splicing map.Why aretheexonicsequencesignored in theRNA map (figure3d)?This makesnosenseconsideringthatmostoftheRBM10bindingclustersarelocatedinexons(figure 2b). 7.Minigeneexperiments(figure4). There are multiple issues with the data presented on this figure: (i) Disrupting the binding sites does significantly disruptthe effectof RBM10 on splicing,possible withtheexceptionofMutD5.Thisisaverystrongargumentthattheauthorsdidnotidentify the correctbinding sites.Thisdata notonly doesnot"provide unequivocalsupport",butdirectly contradictsthe authorsconclusion that"...RBM10 binding in the vicinity ofsplice sitesofflanking introns would facilitate the skipping of the cassette exons". (ii) While the RBM10 effecton PUF60 splicing is mostly abolished in PUF60 MutD5,this is by no meansconclusiveevidencethatthesitedisruptedinMutD5isthecis-acting elementthatis recognized by RBM10.The mutation may have disrupted sequence element recognized by a differentprotein thatisrequired forregulation ofthealternativeexon.Theauthorsneed to show that placing theRBM10 binding sitesin vicinity ofaheterogonousalternativeexon confersregulation by RBM10. (iii) Thereisnostatisticalanalysistoshow how significantthechangesinexoninclusionareand whatisthevariabilityoftheassay. (iv) The authors need to show the sequences of the wild type and mutated binding sites. (v) the Authors need to show gelimagesofthe RT-PCR reactions. 8.Theauthorsconvincingly show theeffectofRBM10 mutationsin patientson alternativesplicing. HoweverRNA bindingproteinsfrequentlymultitaskandregulateRNA stabilityandtranslation.It wouldbeinterestingtoknow if there are transcripts with altered abundance and the patient EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 7 lymphoblasts and after the RBM10 knockdown in HEK cells. Also does RBM10 bind to the mRNA UTRs,whicharefrequentlyinvolvedinregulationoftranslationandRNA stability.Theanswersto these questions should already be in the PAR-CLIPandRNA-Seqdata.Thefactthatthetwo patientspresented in thisstudy display milderphenotypescompared to thetypicalTARP syndrome, despitehaving impaired function ofRBM10 in slicing would arguethattheprotein may have additionalfunctionsin the cytoplasm. Minorissues: 1.Figure1b.From themethodsitisunclearwhy thevaluesfortheconsensusclustersarehigher than the values for the reads. If clusters are aggregates of reads, one would expect the cluster density to be less than the read density. 2.On Figure2ctheauthorsneed to definewhatisconsidered to be"Strong","Medium"and "Weak" binding site. 3.On Figure3cshowing thealigned PAR-CLIPreads,ratherthanatrianglewillbemore informative to the reader. Novelty AlthoughassociationwiththespliceosomeraisesthepossibilitythatRBM10regulatesalternative splicing this has notbeen shown to date.Furthermore,the rich sequence data obtained in this study can provide significant insight into the mechanisms by which RBM10 regulates splicing. MedicalImpact MutationsinRBM10havebeenassociatedwithdevelopmentaldisorders.Furthermore,itis frequently mutated in certain types of cancer.Understanding its function may contributeto developing cancertherapiesand prognosticmarkers. Adequacyofthemodelsystem TheauthorsexpressepitopetaggedRBM10forthePAR-CLIPexperiments.Whilethisis acceptable,particularly in caseswhere good quality antibodiesare notimmediately available for the endogenousprotein,the authorsneed to show thatthe levelsofthe expressed protein are comparable to those of the endogenous protein. Maintaining physiological protein levels is critical as over- expression may resultin binding to low affinity siteson theRNA thatarenotoccupied undernormal conditions. Referee#3(Remarks): RBM10isanRNA bindingproteinthathasbeenassociatedinseveralstudieswiththespliceosome. However,itsfunctiontherehasremainedunclear.Thework presented by Wang etalin this manuscriptascribesafunctionofRBM10insplicingandmorespecificallyinexonrecognition.The authorsuse a combination ofPAR-CLIPandRNA-seq to build an integrated modelforRNA splicing regulation by RBM10.Wang et al also show that alternative splicing patterns in patients withTARPsyndromeresemblethoseofRBM10knockoutcelllines,concludingthatthesplicing regulatory function of RBM10 is disrupted in the patients.The results of the presented work can potentially haveasignificantimpacton ourunderstanding ofsplicing regulation in organism developmentand human disordersincluding cancer. Whilethequalityoftherawdataappearstobeadequatethesubsequentanalysisleavesalottobe desired (seethe specific comments fordetails).The results as presented in the manuscriptdo not supportthe proposed RBM10 binding site sequence and the modelforsplicing regulation by RBM10.Therearealsosomeissuesthatneedtobeaddressedinrespecttothemodel system.In particular,theuseofprotein over-expression in the PAR-CLIPexperimentsmayresultinthe identification of binding sites that are not normally occupied by RBM10. If these deficiencies are adequately addressed the work by Wang etalwillwithoutdoubthavesignificantimpact. 1stRevision - authors'response 07 June 2013 EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 8 Wewouldliketothankthethreerefereesforcarefullyreviewingourmanuscriptandappreciatethe constructive critique raised by the referees. In what follows, we address all aspects that required correction orclarification. Summaryofimportantchangesintherevision: 1. Following the suggestion of all three referees, we asked Dr. Dagmar Wieczorek, the clinical geneticist,who hasbeen taking careofthefamily,to add the clinicaldata and provide heropinion on thegenesregulated by RBM10.In short,weadded adetailed casereportoftheaffected cousins (see Supplementary Information) and a table summarizing the phenotype comparison between our patients and TARP patients reported before (Supplementary table 7). We added a chapter concerning the relevance ofthe genesregulated by RBM10 in causing phenotypesoverlapped with ourpatientsand/orTARP patientsin thediscussion part. 2.Following thesuggestion ofreferee #2 and referee #3,we performed furtheranalysis of ourPAR- CLIPandRNA-seq data.In the revised manuscript,we added detailed clarification aboutRBM10 binding clusters, such as the distribution of length, number of PAR-CLIP reads and etc. We incorporated theappropriatecontroland statisticalanalysiswhen necessary.Weadded achapterand a table (supplementary table 3)listing the genesdifferentially expressed upon RBM10 perturbation. 3. Following the suggestion of referee #2 and referee #3, we performed two additional minigene experimentsin a heterologouscontext.1)weinserted RBM10 binding sitesinto introniclocation of a new splicing reporter, pZW2C;2)we fused RBM10 with a modified pumilio domain,PUF3-2, whichspecifically recognizesan eight-nt sequence 'UGUAUGUA'with high affinity, thereby,we could tether RBM10 close to (18nt downstream) splicing sites of the cassette exon in another splicing reporter. Both minigenes could demonstrate that intronic binding of RBM10 near splice sitesindeed could enhance exon skipping in a heterologouscontext. 4. Following the suggestion of referee #2, we re-organized the discussion part and moved the discussion ofRBM10 exonic binding to the resultpart. We believe that our additional experiments and computational analysis helped to substantially improve thequality of thepaperand to address therequestsmadeby thereferees.Weadded two new authorswho havebeen involved substantially in the revision. Pleasefindanexhaustivepoint-by-point response below. We are grateful that you can consider the manuscriptforyourjournal. Referee#1(Remarks): Thisverywellwrittenand illustratedpaperdescribes theeffectofRBM10onsplicingofcassette exonsand identifiesa numberofgenesdifferentially affected by overexpression or knock down of RBM10.Thelaboratorystudiesarenovelandveryrelevant. A weaknessofthispaperisthelackofcompletemedicalcontext.Whilethereisabriefmentionof the relevance to cancer, and the cancer genesarehighlighted in thesupplementarytable,themore relevant primary context of embryologic development is not appropriately addressed. This major weakness should be addressed by asking the clinicians, who identified the patients on whom this workis based,to contribute to the written manuscript. Wethanktherefereeforhis/herappreciationofthenoveltyandrelevanceofourstudy,andthank him/her and other referees for pointing out the weakness of our manuscript. Following his/her suggestion,weaskedDr.DagmarWieczorek,theclinicalgeneticist,whohasbeentakingcareofthe family,to add the clinicaldata and provide heropinion on the genes regulated by RBM10. In the introduction you state the "100% pre- orpostnatallethalityin affected males". This is wrong, aslong term survivalhasbeen reported {Gripp etal.,American JournalofMedicalGenetics2011}. EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 9 We thank the reviewer for this comment: All but one patient described before died pre- or post- natally. We corrected this in the revision. Indeed, the two cousins described here are the eldest individualswith TARP syndromeso far. Thedescriptionofthepatientfrom whom thecelllinewasderivedisinsufficient.Isthispatientalive or deceased? Is the cousin indicated on the pedigree alive or deceased? What are the typical malformationsseeninTARP,andwhatmajormalformationswerepresentinthepatientwhosecells you used? We added detailed case report of the cousins (see Supplementary Information) and a table summarizing the phenotype comparison between our patients and TARP patients reported before (Supplementary table 7). The index patient, from whom the cell line was available, deceased, whereastheyoungercousinisstillalive.Weupdatethepedigreetomakethisinformation clearer for the reader (see Figure 7A). In the Supplementary Information and supplementary table 7, we havelisted allthemajormalformationspresentin reported TARP patientsand/orourtwo patients. It is clear from the data presented here thatRBM10 affects splicing ofnumerousgenes.Some of these genes listed in the supplementary table (CASK; TBX3; CREBBP; FANCA; POMT1 to name a few obvious examples) are known to be causally involved in congenital anomalies. This should be discussed.Thegenesknown to be associated with congenitalanomaliesshould be highlighted in the table (in addition to cancer- associated genes).Can anyofthesegenes,byvirtueoftheirabnormal splicing,resultin the malformations seen in TARP syndrome? We thank the referee for pointing this out. Following his/her suggestion, we added a chapter concerning thispointto the discussion part.In the chapter,we focused on thegenesimplicated in the TARP syndrome associated anomalies and therefore did not discuss TBX3,CREBBP,FANCA and POMT1 because the reported phenotype caused by themutation identified in these four genes did not overlap with the anomalies in TARP syndrome: TBX3 mutations are causative for ulnar mammary syndrome, which is characterized by posterior limb deficiencies and mammary gland hypoplasiaamongst others.Both main clinical findingswerenotobserved in the individualswith TARP syndrome. Mutations in the CREBBP gene cause Rubinstein-Taybi syndrome, which is a recognizable multiple congenital anomaly syndrome with intellectual disability (ID). The facial features and the broad thumbs and toes are very characteristic and differentfrom TARP syndrome. FANCAis one of the genes associated with Fanconi anemia. There are overlapping clinical findings (pre- and postnatal growth failure, internal malformations, hearing loss), but radial or thumb anomaliesand early bone marrow failure,two majorfeaturesofFanconia anemia,were notreported in the individuals with TARP syndrome. Dystroglycanopathies are caused by mutations of the POMT1 gene. The clinical spectrum of anomalies is wide and thus overlapping with TARP syndrome. As results of muscle biopsies were not reported in the TARP individuals, one cannot exclude thatthere mightbe dystrophic changes, but the pattern of malformations in TARP syndrome seems to be differentfrom the POMT1related disorders,e.g.Walker-Warburgsyndrome. WhileIrealizethatthisisnotthefocusofthisparticularreport,thisisthecontextinwhichthework is relevantto human developmentand medicine.A clinicians'inputin thismanuscriptisnecessary. Dr.DagmarWieczorek, theclinicalgeneticist,whohasbeen takingcareof thefamily,added the clinicaldata and provided heropinion on thegenesregulated by RBM10. Minorissues: page8:"anti-correlation"- do you mean inversecorrelation? Yes.Wereplacedtheterm. Theterm "mentalretardation"isnotusedinprofessionalpublicationsanylonger;use"intellectual disability"instead. Thanksforpointing thisout,wecorrected it. DefineTARPatitsfirstuse.Whatdothelettersstandfor?EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 10 TARP stands for Talipes equinovarus, Atrial septal defect, Robin sequence, and Persistent left superiorvena cava.We now added the definition atits firstappearancein themanuscript. page14;middleparagraph:thesentence"Thissuggested thismotif..."doesnotmakesenseas written. Wechangedthesentencenowto“Thissuggestedthemotifmightnotrepresentthespecific sequencesrecognized by RBM10”. "Acknowledgement"ismisspelled. Thanksforpointingthisout,wecorrectedit. Referee#2(Remarks): In the manuscript entitled "Integrative analysis revealed the molecular mechanism underlying RBM10-mediated splicing regulation" Wang et al. perform PAR-CLIP to identify genome-wide binding sites for the RBM10 protein, and perform RBM10 KD and overexpression experiments followed by RNA-seq analysis to identify RBM10 dependentsplicing changes.The authors then use the genomics data to try to characterize the mechanisms of RBM10 action based on correlative analyses between RBM10 binding and alternative isoform usage. Finally, the authors perform a limited analysis of an apparent NLS deletion RBM10 mutation associated with human TARP syndrome, finding that this patient sample shares the alternative splicing profile of the RBM10 knockdown. Althoughseeminglypromising,themanuscriptlacksaclearandcompellingbiologicalstorythatis supported with solid evidence,and seems to be missing many more detailed analyses to make the results more convincing. The CLIP and RNA-seq experiments will provide datasets for further research on RBM10, and the proposed link between RBM10 and the U2 snRNP is interesting (although notverywellexplored experimentally).However,the RBM10 binding mechanism remains unclear (asno motif is identified from the intronicbinding sites,and theGAAGA motif identified from exonic binding sites apparently didn't validate in gel shift assays), and the effect of RBM10 binding,even in the limited minigene assays presented, is weak (~5% effect on exon inclusion). In addition, while the majority of the manuscript focuses on intronic RBM10 binding, the profile in Figure2aissignificantlyshiftedtowardsexonicbinding(~40% exonicvs50% intronic) as opposed to the whole human genome (less than 1:5 exonic to intronic), suggesting that the exonicbinding sites are more likely to be biologically relevant. Similarly, the TARP syndrome section feels a bit tacked on; it is interesting (though perhaps not surprising)thata patientsample with an RBM10 NLS deletion would resemble (atthe splicing level) RBM10 knockdown, but it remains unclear to what degree this altered splicing pattern actually leads to phenotypic effects associated with the disease.As theauthorsdon'tdiscuss thedegree to whichgeneexpressionisalteredinthesesamples,it'snotclearwhetheralteredgeneexpressionor altered splicing isthemajorcomponentofTARP syndromein thisindividual. Asageneralcomment,the supplementalfiguresneed to be higherresolution (asthey're impossible to read as presented here). Followingthesuggestionofthereferee,weperformedfurtheranalysisofourPAR-CLIPandRNA- seq data.In the revised manuscript,we added detailed clarification about RBM10 binding clusters. Weincorporatedtheappropriatecontrolandstatisticalanalysiswhennecessary.Weaddedachapter and a table (supplementary table 3) listing the genes differentially expressed upon RBM10 perturbation. In addition, to validate the impact of RBM10 binding on alternative splicing, we performed two additional minigene experiments in a heterologous context. Both minigenes could demonstrate thatintronic binding ofRBM10 nearsplice sites indeed could enhance exon skipping in a heterologous context. We admit that we did not detect sequence motif directly recognized by RBM10. However, given that many protein interaction partners of RBM10 are RNA bindingEMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 11 proteins(seeHegeleA,et.al.Molecularcell45:567-580),itisplausible that RBM10 binds to RNA also indirectly via other interaction partners. Weagreewiththereferee,itappearedthatRBM10bindingclustersweremoreenrichedinexons than in introns (see also the table presented in the detailed point-by-point response below). However,please note the numberofnucleotidesin the genome corresponding to exonic orintronic regions was used as the background to calculate the enrichment.Given thatthe percentage of introns and exonsrepresented in cellularRNA isvery differentfrom thatin the genome,(i.e.the majority of cellularRNA derived from protein-coding genesare mature mRNAscontaining no introns),and that the IP was performed on RNA instead of genomic DNA, such enrichment value should be treated morecautiously.Asto the potential function of exonic binding, we observed that RBM10 binding at exonic GAAGA sitesappeared to override the exon skipping effectofintronic binding.Exceptthis, we tried, but failed to find any additional clear correlation between exonic binding and splicing changesinduced by RBM10 perturbations.Comparing with the neighboring constitutive exons,the density of binding sites in the cassette exonswas reduced.Thiswasobserved for all the cassette exons,the exonsmore excluded upon RBM10 OE,aswellasthe exonsmore included upon RBM10 OE, andcouldprobablybe explainedby the fact that cassette exons are non-constitutive and are therefore not always present in all transcripts. The fact that there is no clear difference in exon binding between differentgroupsofexonsindicatesthatthe exon binding alone mightnotregulate splicing.Having said these,we could notexclude the effectofexonic binding on otherprocesses such as mRNA translation,which is beyond the scope ofthis study. In the revised manuscript, we added a detailed case report of the affected cousins (see Supplementary Information) and a table summarizing the phenotype comparison between our patients and TARP patients reported before (Supplementary table 7). We added a chapter concerning the relevanceofthegenesregulated by RBM10 in causing phenotypesoverlapped with ourpatientsand/orTARP patientsin thediscussion part. Weincreasedtheresolutionofallsupplementaryfigures,whicharenowinvector-based format. We believe that our revision substantially improved the quality of the paper and addressed the requests made by the referee.Please find an exhaustive point-by-pointresponsebelow. Moredetailedcommentsarebelow: PAR-CLIPanalysis: Forthebindingnearexons(Fig2b),thereseemstobeacontrollinemissing.A shuffledcontrolor even anotherprotein's PAR-CLIPdatasetwouldmaketheresultsforRBM10moreconvincing. WenowaddedAGO2PAR-CLIPdataset(thesamePAR-CLIPexperimentalcondition in thesame cellline,i.e.HEK293)ascontrol.Asshown in Figure 2,compared with AGO2,RBM10 binding shows clearenrichmentin the vicinity ofboth 5’and 3’splicing sites. In Figure 1a,I assume thatUTRs are included in the exon annotations- whatdoesthedistribution look like if you separate them from exons? Is there any UTR enrichment? Wenowchanged thepie chart ofgenomicdistributionofRBM10bindingclusters, inwhich the exonswere separated into coding sequence,5’and 3’UTR(seeFig.2A).Comparingwithcoding exonsand the splicing sites,both 5’and 3’UTR show much lessenrichment,see the table below, RBM10BindingSites Nucleotides FoldEnrichment Exons 34,426 (39.1%) 70,480,509 (2.5%) 15.87x 5'UTR 921 (1.0%) 11,843,639 (0.4%) 2.53x CDS 30,327 (34.5%) 34,759,483 (1.2%) 28.36x 3'UTR 3,178 (3.6%) 23,877,387 (0.8%) 4.33x Introns 45,673 (51.9%) 1,006,012,043 (35.2%) 1.48x EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 12 5'SS 8,340 (9.5%) 26,221,461 (0.9%) 10.34x Intron Center 25,170 (28.6%) 953,551,249 (33.4%) 0.86x 3'SS 12,163 (13.8%) 26,239,333 (0.9%) 15.07x Intergenic 7,858 (8.9%) 1,782,182,170 (62.3%) 0.14x Total 87,957 (100.0%) 2,858,674,722 (100.0%) 1.00x Pleasenoteweusedthenumberofnucleotides in thegenomecorrespondingtodifferentgenomic regions as the background to calculate the enrichment. As explained above, given that the percentageoftheseregionsrepresented in cellular RNA is very different from that in the genome, such enrichmentvalue should be treated more cautiously. Themotifanalysisseemstohavebeendoneinaveryspecificwayinordertoacquiresomesortof enrichment. Imissed any description of the algorithm used formotif finding other than 'pentamer enrichment' - was the GAAGA motif most significantly enriched, greatest fold-enriched, etc? A statisticaltestshould also be included for significance ofenrichmentinexonsandinthevicinityof both 5'and 3' splice sites of the introns. The authors also do not describe their definition of strong/medium/weak binding sites. I'm also confused by the apparent periodicity of pentamer frequenciesin 2c- is there an explanation for why this might be observed? Oristhisan artifactof some unspecified windowing or normalizationprocedure? The motif analysis was done in a very simple and straightforward way. We determined the frequency of pentamers inside 40-nt windows around preferred crosslinking sites and compared them to the frequency around control sites. There is no clear sequence motif enriched in the intronic binding sites.Themostabundantpentameraround RBM10 exonicbinding siteswasGAAGA,e.g. 25.3% ofallRBM10 siteshaveaGAAGA motifin up to 20bp distance,whereasonly 10.8% ofthe controlsiteshave a GAAGA motif in up to 20bp distance (Fisherexact testp-value< 10-16).As shown in the figure below,othermotifs with high enrichmentare justslightdeviants ofGAAGA. 00 .0 2 0. 04 0. 06 0. 08 0. 1 0. 12 .020.40.60.80.10.120.140.160.180.20.20.240.260.28 Bac kg ro und Fr eq ue nc y Frequency at Exonic RBM10 Binding Sites GAAGA AAGAA AGAAG TGAAGAGAAA AAGAT Wenowaddedthedefinition ofstrong/medium/weak binding sitesin themethod section.In brief, we sorted the binding clusters according to the number of reads spanning the binding site. The clusters were then binned into three groups of equal size. Theperiodicityofpentamerfrequenciesisdueto codon usageand thefactthatwerequired aT at position 0. Fig2dseemsunnecessaryasamainfigureorevenasupplementalfigure,asitisaveryweakresult. WethinkFig.2Dshowedthatalthoughthereisnosequencemotif for intronic binding, there is still clearbiasin nucleotide composition. Interestingly, such bias is similar between the binding at 5’ and EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 13 3’splicing sites,although thosesiteshavequitedifferentbackground basecomposition,indicating the bias may representthe sequence preference ofRBM10 binding. Thediscussionincludesmentionofagel-shiftassay thatfailed to detectdirectbinding ofRBM10 to the GAAGA motif described in figure 2c. This result needs to be presented in the results section during thediscussion offigure2c,asithassignificantimplicationsforinterpretation offigure2c.It also needs to be further discussed, as at the end of the paper it is unclear whether the authors believe that this reflects an RBM10 motif or rather amotifofsomeunspecifiedadditionalregulator that RBM10 associates with. As the referee suggested, we now presented the gel shift assay (Supplementary Fig 1.G) and discussed the relevance of this finding in the result part, immediately following the motif identification part. We make it clear that we believe that RBM10 binds to the motif via other interaction partner(s). TheinferenceofU2snRNA bindingbyPAR-CLIPreaddensityinFigure2eisinteresting- for the second partoffigure 2e,itwould behelpfulto includethedata (read counts/base),asit'sunclear whetherthetwoindicatedpositionsaremostenrichedoraretheonlypositions observed to have the T->C crosslinktransitions. Astherefereesuggested,weincludednow anotherfigure(Fig.2E,up rightpanel) for the density of T-C conversionreadsalongU2.Asshownintheplot,thereareclearlytwocrosslinksiteswithmost enriched T-C conversions,indicated in the figure below (Fig.2E,low panel). RBM10OE/KD RNA-seq: The authors go straight to altered splicing upon RBM10 KD and OE - are there significant alterations at thegeneexpression level? I recognize that the focus of thispaper is on alternative splicing regulation by RBM10,but the scale to which RBM10 manipulation generallyeffectsgene expression would be valuable (aswellas information on otherRNA binding proteins significantly altered in theRBM10 KD/OE experiments). WenowaddedthefollowingparagraphdescribingthegeneexpressionchangesinducedbyRBM10 OE/KD in the resultpart. “Thegeneexpression levelwasestimated based on RPKM (readsperkilobaseofexon permillion mapped sequence reads, (Mortazavi et al., 2008), Material and Methods). At false discovery rate (fdr) < 0.05,171 and 105 genes were found to besignificantly upregulated and downregulated by at least 1.5 fold upon RBM10 KD (Figure S2F and Table S3), whereas 19 and 49 genes were upregulated and downregulated to the same level (fdr < 0.05, fold change ≥ 1.5) in response to RBM10OE,respectively (Figure S2F and Table S3). Notably, the expression changes induced by KD andOEwerenotinverselycorrelated(FigureS2G).” AmongthegenedifferentiallyexpressedunderRBM10OE/KD,14wereRNA-binding proteins and five were known to be splicing regulators, which might have secondary effect on the splicing changesinduced by RBM10 perturbation.We added thispointin the resultpart. In SupplementalFig.2a,the western blotfor the RBM10 KD and controlsample needs to be shown together on the samewesternblotasdoneinSuppFig2b.Asitis,itappearsthattheauthorspasted together two lanes from potentially different gels, which may or may not show an actual KD at the protein level.Quantification (particularlyoftheknockdown western blot) would also be ideal,as it appearsthatGAPDH isalso lowerin theKD sample. WenowincludedtheoriginalwesternblotinSuppFig2andshowedthechangeattheproteinlevel based on quantification of the signal intensity from the specific band (normalized to the level of GAPDH). Itseems thatthe overlap ofevents changed in the OE and the KD is 306,butwhataboutthe events that do not overlap (not in both the OE and KD experiment)- is there anything interesting with those? Are these just false positivesorweretheyjustmissed in oneoftheexperiments? Can theybe validated in both conditions? EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 14 Webelieve the splicingchanges thatwereonlyobserved ineitherOEorKD,or those thatwere positively correlated between OE and KD,are trueevents. The major reasons for such a behavior include 1) The basal level of the exon inclusion in unperturbed HEK293 cellsistoo high ortoo low. In the former situation, the change induced by OE, i.e. exon skipping, could be very obvious whereas KD could hardly produce any further/additional exon inclusion. The opposite situation holdsalso trueforthoseexonswith too low basallevelofexon inclusion.Asshown in thefigure below, compared with those exons showing splicing changes in both conditions, the exons with changes in only one condition have more extreme basal level of exon inclusion. 2) Those events represent secondary effects, which could result from the expression change of certain splicing regulators.As discussed above, the expression changes were not inversely correlated between KD and OE. -0 .2 -0 .1 0.0 0.1 0.2 0.3 0.4 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 ∆PSI in OE ∆PSI in KD 0.2 ≤PSI Ctrl ≤ 0.9 PSI Ctrl > 0.9 PSI Ctrl < 0.2 Todemonstratesuchchangeswerenotfalsepositive,usingqPCR,wevalidatedthesplicingchanges of four exons, which are positively correlated between OE and KD (see Fig 3A-B and supplementaryFig.3). Theauthorsreport17eventsthatvalidatedviaqPCR - however,itisdifficultto determinefrom the data shown how representative these events are, as 11 of the 17 have >~2-fold changes in PSO whereas themajorityof events in figure3ahavedeltaPSI<|0.2|. Additionally, detecting splicing changesby qPCR requiresa fairamountofnormalization calculations, but these validations can be donemoresimplywith RT-PCR andrunningagarosegels.Canthesplicingchangesbedetectedthis wayorweretheyonlydetectablebyqPCR? Wenowmarkedthe21eventsthatwerevalidatedbyusingqPCRinFig.3A.Asshownthere,these eventscould representthe events induced by OE and KD.As mentioned above,we now validated the splicing changes of four exons, which are positively correlated between OE and KD.To assure that the splicing changes could be detected via both qPCR and normal RT-PCR followedbyrunning AgilentBioanalyzer,wevalidatedthesplicing changes of 8 exons also using the latter approach. As shown in thefigurebelow,thechangesmeasured by both approachescorrelated well. EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 15 R "="0.788 0" 0.5" 1" 1.5" 2" 2.5" 0" 0.5" 1" 1.5" 2" qPCR"log2FC(PSO(OE"vs."Ctrl))" RT= onesplicing sitesshowed splicing changeatthelevelofZ<=-2 and Z<=-1.Therefore, it is clear that exons with RBM10 binding showed stronger skipping upon RBM10 OE. However, given thecomplicatesplicing regulatory network,itisconceivable notallthe binding eventscould lead to significant splicing changes. The similar phenomenon is often observed in the study of transcription factors. Ontheotherhand,therearein total1477 and 3122 exonswith splicing changeatthelevelofZ<=-2 and Z<=-1,respectively, of which 455 (30.8%) and 878 (28.1%) were associated with at least one RBM10bindingatthesplicingsitesofadjacentintrons.TheremainingexonswithoutsuchRBM10 binding sites could well represent the secondary effect given that a number of known splicing regulators were differentially expressed/spliced upon RBM10 perturbation. Finally,wedetectedintotal412exonswithsignificantsplicing changes(fdr< 0.05,|∆PSI|≥ 10%), of which 127 (30.8%) were associated with at least one RBM10 binding at the splicing sites of adjacentintrons.Again,the remaining onescould wellrepresentthe secondary effect. RBM10splicingmodels: Theminigeneexperimentsseem likeaniceidea,butIunfortunatelyIdon'tthinktheresultsareas convincing asclaimed.In Figure 4,the authorsstate that"These data provide unequivocalsupport to our hypothesis that RBM10 binding ... would facilitate the skipping of cassette exons", but the lack of technical qPCR error bars as well as some sort of statistical test to show whether the observed changes are significantconflictwith such a strong statement. In Fig.4B and C,we showed the results from the three replicates.Using two tailedpaired t test, comparing the RBM10 induced splicing changes between the minigenes containing wild-type or mutantRBM10-binding sites,theobserved differences(exceptDel_U3 forPUF60),although subtle in absolute values,are indeed statistically significant.We now added the p-value to the legend of Fig.4B andC. Additionally, the effect on splicing for three of the four mutations made in Fig 4b/c is extremely small(~5% or less),and the data shown only reflects the change in exon exclusion between OE and control.Iwascuriousasto whetherthe mutationsperformed show significantly altered PSO rates in the control (or over-expression)by themselves,and to whatthe ratesofexon inclusion forthese EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 17 eventstypically are (i.e., if they were 90% excluded in the control then a small effect upon RBM10 OEwouldbenotsurprising). Since the binding sites are close to the splicing site, unsurprisingly, once mutated, the splicing pattern even in thecontrol was changed (see the supp Fig.5).The PSO rates in the minigene without RBM10OErangedfrom 30% to90%. Additionally, it would be ideal to perform the direct experiment (forced RBM10 association with intronic loci, e.g. by MS2 tagging) to prove that RBM10 recruitment to an intron willaltersplice site choice. We thank the referee for thesuggestion.To further test theeffectofRBM10 intronicbinding on exon skipping,we performed two additionalminigene experimentsin a heterologouscontext. First,we insertedRBM10bindingsites into intronic locationofanew splicingreporter,pZW2C, whichwasconstructedbyinsertingexon2oftheChinesehamsterdihydrofolatereductasegeneand part of its flanking intronsbetween two GFP exons (Figure4D) (Wang etal,2013).We inserted three RBM10 binding sites from PUF60and assayed the exon skipping changesupon RBM10 OE respectively. As shown in Figure 4E, insertion of all the three RBM10 binding sites exhibited significantly strongerskipping effects upon RBM10 OE compared with thecontrol,demonstrating that intronic binding of RBM10 near splice sites indeed could enhance exon skipping in a heterologouscontext. Second,astherefereesuggested,wefusedRBM10withamodifiedpumiliodomain,PUF3-2,which specifically recognizesan 8-ntsequence'UGUAUGUA'with high affinity (Figure4F)(Wang etal, 2009).Thereby,wecould tetherRBM10 close to (18ntdownstream)splicing sites of the cassette exon (Figure 4F) (Wang et al, 2009). As shown in Figure 4G, the expression of RBM10-PUF induced strong exon skipping effects, while expression of PUF3-2 alone showed hardly any changes. Webelievedthethreeminigeneexperimentstogethernowcouldprovideunequivocalsupportto our hypothesis that RBM10 binding in the vicinity of splicing sites would facilitate the skipping of cassette exons. P-valuesare missing forFig.5d.The strength ofsplicing sitesdistalto the cassette exonsdoesnot seem significantly stronger than that of those immediately flanking the exons, and in general the effectsize here seemsto be very slight. Weagreethattheeffectlooksquiteslight,however,thestrengthsofdistal5’(3’)splicingsitesis significantly higher than the strengths of5’ (3’, respectively) splicing sites directly flanking non- constitutive cassette exons(KS-test, p≈0). Furthermore, in Fig. 5C, (previously Fig. 5D), we want to stress that the difference is also statistically significant between the splicing sites immediately flanking all cassette exons and those with higher exclusion upon RBM10 OE.We added these p- valuesto thefigure. Itlooks like fig 6b should be recreated by the authors,seems like itwas taken from another publication. Fig.6B wasindeedcreatedbyYongboWangbasedonUniprotandPfam proteindomain annotation using asoftwarecalled “DOG1.0”(http://dog.biocuckoo.org/). Nevertheless,to avoid any possible misunderstanding,wereplacedwithanewfigure. TARP syndromeanalysis: Intriguingly,the splicing changes observed here correlated wellwith changes induced by RBM10 KD inHEK293(Fig.6c).WhataboutcomparedtotheOEexperiment? BothRBM10KD andthemutantRBM10identifiedinthepatientrepresentloss-of-function events. Thereforewecompared in themanuscriptonly between thepatient and RBM10 KD. As shown in EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 18 the figure below, the changes identified in the patient were inversely correlated with OE.But, as expected,the correlation coefficientismuch lowerin thiscase (R2: 0.177 for KD vs0.0252 forOE). R²= 0.0252 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 ΔPS Iin LC LP at ien tv s. No rm al ΔPSI inHEK RBM10OE Supplemental figure 3 needs better quantification - it is clear from the figure that the mutated RBM10isnotsilenced,butitisdifficulttotellwhethertheexpressionisactuallyunchanged from the results shown. Wequantifiedtheexpressiondifferencebasedonthesignalintensity oftheband on western blot and now included the numberin the supp fig 6. Figure6calsoneedsstatistics- whatisthecorrelationbetweenthetwosamples? WeincludednowthecorrelationcoefficientforFigure7C(previouslyFigure6C). Figure6c-e are used to propose thatthispatientdeletion actsasan RBM10 deletion through lossof nuclearexpression.However,thedeletion includesnotonlytheNLS butalso additionalsequences. Figure6ewouldbestrengthenedbymutatingeitheronlythe NLS,orattachinganormalNLStothe mutatedRBM10toshowthatthemolecularphenotypes observed in this patient are characteristic mis-localization of RBM10 and not also loss of function through deletion of additional domains. Thedeletionremovedalso otherfunctionaldomains,including a zinc fingerdomain and partofa G patch domain.However,asshown in Figure7B,thelossofNLS willabolish thenuclearfunction regardless of the effecton other domains.In this manuscript,we focused on the splicing regulation mediatedbyRBM10andclearlyshowedthatthemutantidentifiedinthepatientlostsuchfunction dueto mis-localization, and the splicing changes correlated wellwith the changes upon RBM10 KD. Discussioncomments: Overall,thediscussion needs to be rewritten to put the results of the paper in context. The idea the RBM10 works with other RBPs needs to be presented sooner than the discussion, otherwise the results section makes no sense in this context - for example, it is not until the discussion that the authors imply that RBM10 does not seem to bind the identified motifs by itself. EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 19 Astherefereesuggested,wemovedthediscussionofRBM10exonicbindingintotheresultpart. Similarly,theTARP section needsa betterdescription of how it fits with the results in the rest of the paper- if RBM10 loss of function causing TARP is previously known, is the novelty the validation of the individual deletion patient as actually losing nuclear localization? Or that the loss of nuclear localization of RBM10 resembles RBM10 knockdown? In previous findings,only nonsense or frameshiftmutations in RBM10 were identified in the TARP patient.Weforthefirsttimeidentified an in-frame deletion and showed showed the loss of nuclear localization ofRBM10 could lead to asimilarphenotype,thereby demonstrating thatRBM10 exerts a criticalfunction in the cellnucleusduring development.Atmolecularlevel,we also showed that mis-localized RMB10 mutant protein renders similar splicing de-regulation as RBM10 KD, supporting again thatRBM10 predominantly functions in the nucleus as a novel splicing regulator. Thestatement"Whole-mount insituexpressionanalysisof themurineRbm10hasshown that the gene was expressed during embryonic development in a pattern consistent with the human malformationsobservedinTARPsyndrome"ismissingareference. Weaddednowthereference. Thediscussionreferstotwoexperimentsthatneedtobeincorporatedintothemaintext,astheyare not mentioned anywhere in the manuscript before the discussion - one experiment (an in vitro binding RBM10 experiment) that is not shown in any figures and only mentioned in the supplemental methods, and discussion of the effect of exonic RBM10 binding (figure 7), which is also notpresented in the results. Astherefereesuggested,weincludedthegelshiftassayinthesupplementaryfigure1andeffectof exonic RBM10 binding in the resultpart. Minorcomments: -Breitlingetal2004citationinthemethodssectionis misformatted and notincluded in reference list Thanksforpointingthisout,wecorrectedit. -typo in figure reference: "In agreement with our in vivo data, the skipping of the cassette exons was enhanced upon RBM10 OE (Fig.54b)" Thanksforpointing this out, we corrected it. -Figure5figurelegendismislabeledasfigure4 Thanksforpointingthisout,wecorrectedit. Referee#3(CommentsonNovelty/ModelSystem): Technicalquality Majorissues: 1.Filtering outpotentialcontaminants.Whilemostofthereadsarederivedfromcross-linked RNA species (carry U to C transitions ,from supplementary figure 1a itis clear thatthere is successful RNA pull-down from thecontrolsamplesthatwerenotcross-linked. Such result indicates that the washingstringencymaynothavebeensufficienttoremovespeciesofRNAretainednon-specifically on the resin or bound through mediated interaction. Reads derived from such non-specifically interacting RNAs can clearly be identified in PAR-CLIPbytheabsenceofU to C transition in the sequence.Werethosereadsremoved from thesetthatwasused to definetheRBM10 binding site? Notall theUs inallRNAscouldbe labeledwith4sU,notallproteins interacted i4sUscouldbe crosslinked and not all crosslinked 4sUs will be converted to C during RT-PCR. Therefore, to achieve an optimal balance between sensitivity and specificity, the in-house PAR-CLIP dataEMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 20 analysispipeline [also used in Lebedeva et.al.2011]used also the readswithoutT to C transition, butrequired each clusterto contain atleastoneread with T to C transition.Asabriefdescription of the pipeline, reads were firstly quality trimmed, adapters were removed. Pre-processsed readswere then mapped to the human reference genome UCSC hg19 withoutseeding,allowing foratmostone mismatch, insertion or deletion (edit distance of 1). Clusters were called on the set of uniquely mappablereadsif1)atleasttworeadssupportthecluster and 2) at least one T->C conversionwas detected within theclustered reads.Thepreferred cross-linked site for each cluster was defined as the site with the highest number of T->C conversionevents.Clusterswerescoredbythenumberof T->C conversions.A false-discovery rateof<= 5% wasobtained by filtering clusters on the quality scores, considering reads mapping antisense to annotations and reads mapping to chrY as false- positives.Asshown in thefigurebelow,theno.ofPAR-CLIPreadsandthereadscontainingT-C conversionswithin each clusterwere highly correlated. 2 4 6 8 10 2 4 6 8 10 Reproducible clusters log2(PAR−CLIP reads per cluster) log2(Con ver ted reads per cluster) 2.Binding siteclusters.Theauthorsneed to providemoreinformation on theclusterthatwere identified in the PAR-CLIPexperiment: (i) Whatis the distribution ofthe cluster sizes? Weincludednowthedistributionofclustersizes in supp figure 1.As shown there,mostwere between 20 and 40 nt. (ii) How many reads are forming a typical cluster and what is the read count distribution of the clusters? The relatively smalloverlap between the two experimentswould indicate that most of the clustersare formed by relatively few reads. WenowincludedthedistributionofPAR-CLIPreadcountsforallclusterortheconsensusclusters in supp figure 1. As expected, it is clear the consensus ones were formed with more PAR-CLIPEMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 21 reads than non-overlapping ones, demonstrating the consensus binding clusters represent more stableand morelikely functionally relevantevents. (iii) Within each cluster,are there preferred cross-linking sites? This would be a strong indication ofthepresence ofhigh affinity binding site. Therearepreferredcross-linking sites within each cluster. See the response above for the brief description ofPAR-CLIPdataanalysis. 3.RBM10 binding sitesequence.TheauthorsidentifyGAAGA asa pentamerthatis enriched in the RBM10bindingsiteclustersthatarelocatedwithinexons.TheabsenceofUridineresiduefrom the binding siteisquitesurprising considering thatPAR-CLIPisrelianton cross-linking to U residues. Itraises the possibility thatthe approach used to identifythebinding sitesequenceisinadequate. In particular whatwas the rationalto analyze separately the exonic and intronic binding sites? Do the authors expect RBM10 to bind to a different sequence in the introns compared to theexons? Was the GAAGA pentamer significantly enriched in the intronic binding sites? If not, than what is the evidence of sequence specific binding of RBM10 to RNA? Alternatively it is possible that the sequence recognized by RBM10 does notcontain uridines thatcan becross-linked to the protein. ThiswouldmeanthatPAR-CLIPisnotanadequateexperimentalapproachforidentifyingRBM10 binding sitesand thestandard CLIP approach,thatusesshortwavelength UV lightto crosslinkto unmodified RNA should beused instead. We agree with the reviewer's view that it seems counter-intuitive to find a U depleted pentamer motif using 4-thiourdine enhanced crosslinking. However in this respect we would like to point out that there is no need for the RNA-recognition element itself to be crosslinked to the protein of interest to capture the RNA target fragment by PAR-CLIP.Hafnerandcolleaguesshowed,intheir originalPAR-CLIPstudy,thatRNA-target sites of the RNA binding protein QUAKING, despite the presence of U in the recognition element, are crosslinked through uridines outside, but close proximity,oftheA(C/U)UAA(C/U)recognition element(Hafneretal.Cell2012 Figure3E+F). We did not expect RBM10 binding differently between exons and intron. Indeed, we started the motif analysis without separating exonic and intronic binding clusters. GAAGA and its close derivativesturned outto theonly enriched motifs, and the enrichment was accounted for totally by the exonic binding clusters and there is no such enrichment in the intronic binding clusters (see also the response to referee #2). As described in the manuscript, we could not demonstrate the in vitro binding ofimmuno-purified RBM10 with an oligoribonucleotidecontaining theGAAGA motifby gel-shift assay although crude HEK 293 cell lysates did bind with the same oligoribonucleotide under the sameexperimentalcondition.This suggested thismotifcould not represent the specific sequences recognized by RBM10. Instead, GAAGA motif is a known binding motif of several serine/arginine-rich (SR) proteins (Long & Caceres, 2009; Sanford et al, 2009). Therefore, it is tempting to speculate that RBM10 was associated with this motif indirectly via other protein partners. 4. Binding to U2. The finding that RBM10 binds to U2 snRNA is quite intriguing. How many reads weremappedtoU2comparedtothereadsmappedinproteincodinggenes.Considering the high abundanceofthesnRNP RNAs,isthereenrichmentofRBM10 cross-links to U2 that is statistically significant? In total, there are 0.25 million RBM10 PAR-CLIP reads mapped to U2, whereas there are 13,51 millionPAR-CLIPreadsmapped(includingbothuniquelyandnon-uniquely mapped reads) to all protein coding genes. However, without precise abundance estimation of U2 versus all protein coding genes, it is difficult to compare the binding of RBM10 on U2 to that on the sum of all protein coding genes.Instead,in orderto estimate the statisticalsignificanceofRBM10-U2interaction,we compared the binding ofRBM10 on U2 with thatofAGO2, in which the PAR-CLIPexperiment wasperformedwith the sameprotocol and in the samecell line. InAGO2PAR-CLIP, only 582 PAR-CLIP reads mapped to U2 and 0.69 million PAR-CLIP reads mapped to all protein coding EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 22 genes.Using Fishertest,wecan demonstratethebinding ofRBM10 on U2 isstatistically significant (P value < 2.2 e-16) 5.Correlation between RBM10 binding and effecton splicing.Onestrong aspectofthepresented work is the availability of both RNA binding (PAR-CLIP) and exon inclusion data (RNA-seq). Surprisingly the authors do not provide information as to what fraction of the RBM10 regulated exons contain RBM10 binding sites. Good correlation between RBM10 binding and exon inclusion/skipping would strongly argue thatthe effecton splicing is specific to RBM10 and is nota secondaryeffect. In total, there are 20,503 binding sites were within 150nt from 5’ or 3’ splicing sites. There are 5,262 non-constitutive cassette exonsassociated with atleastonesuch RBM10 binding sites within adjacentintrons.We detected in total412 exonswith significantsplicing changes(fdr< 0.05,|∆PSI| ≥ 10%),ofwhich127(30.8%)wereassociatedwithRBM10bindingonatleastonesplicing sites in the adjacent introns. The remaining ones could well represent the secondary effect. Seealsothemoreextensiveresponsetoreferee#2. 6.RNA splicing map.Whyaretheexonicsequencesignored in theRNA map (figure3d)? This makesnosenseconsidering that most of the RBM10 binding clusters are located in exons (figure 2b). Asdescribedinthemanuscript,weobservedthatRBM10bindingatexonicGAAGA sitesappeared to override the exon skipping effect of intronic binding. Except this, we tried, but failed to find any additional clear correlation between exonic binding and splicing changes induced by RBM10 perturbations. Comparing with the neighboring constitutive exons, the binding is lower in all the cassette exons,the exons more excluded upon RBM10 OE,aswellastheexonsmoreincluded upon RBM10OE.Thefactthatthereisnocleardifferenceinexonbindingbetweendifferentgroupsof exonsindicatesthatthe exon binding alone mightnotcontribute much to splicing regulation.Having said these, we could not exclude the effect of exonic binding on other processes such as mRNA translation, which is beyond the scope of this study. See also the response to referee #2. 7.Minigeneexperiments(figure4).Therearemultipleissueswith thedata presented on thisfigure: (i) Disrupting the binding sites does significantly disruptthe effectofRBM10 on splicing,possible withtheexceptionofMutD5.Thisisaverystrongargumentthattheauthorsdidnotidentifythe correct binding sites. This data not only does not "provide unequivocal support", but directly contradictsthe authorsconclusion that"...RBM10 binding in the vicinity ofsplice sitesofflanking introns would facilitate the skipping of the cassette exons". Given thatanumber ofgenes differentially expressed/spliced upon RBM10 OE, it is conceivable that the fact that disrupting the binding sites does nottotally abolish the effectof RBM10 on splicing is probably due to the secondary effects,which do notdepend on directRBM10-RNA interaction. Thereforetofurtherproveourhypothesis,weperformedtwoadditionalminigeneexperimentsina heterologouscontext,astherefereesuggested.Seealso theresponseto referee #2 (ii) While the RBM10 effecton PUF60 splicing is mostly abolished in PUF60 Mut D5, this is by no meansconclusiveevidencethatthesitedisruptedinMutD5isthecis-acting elementthatis recognized byRBM10.Themutation mayhavedisrupted sequenceelementrecognized bya different protein that is required forregulation ofthe alternative exon.The authorsneed to show thatplacing the RBM10 binding sites in vicinity of a heterogonous alternative exon confers regulation by RBM10. To validate the impact of RBM10 binding on alternative splicing, we performed two additional minigeneexperimentsinaheterologouscontext.Seethemoreextensiveresponsetoreferee#2. (iii) There is no statisticalanalysis to show how significantthe changes in exon inclusion are and whatisthevariabilityoftheassay. EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 23 In Fig.4B and C,we showed the results from the three replicates.Using two tailed paired t test, comparing the RBM10 induced splicing changes between wild type minigene and minigene containing mutantbinding sites,the observed differences (exceptPUF60 Del_U3),althoughsubtle in absolute values, are indeed statistically significant. We now added the p value to the legend of Fig.4B andC. (iv) The authors need to show the sequences ofthe wild type and mutated binding sites. Welistedthesequencesnow in Supp table6. (v) the Authors need to show gelimages ofthe RT-PCR reactions. WeshowednowbothagaroseandAgilentbioanalyzergel images inSuppfig5 .Giventhevery subtle changes between different conditions, we used the Agilent bioanalyzer for quantification, whichismuchmoresensitiveandaccurate. 8.Theauthorsconvincinglyshow theeffectofRBM10mutationsinpatientsonalternativesplicing. HoweverRNAbinding proteins frequently multitask and regulate RNA stability and translation.It wouldbeinterestingtoknow iftherearetranscriptswithalteredabundanceandthepatient lymphoblastsand aftertheRBM10 knockdown in HEK cells. WenowaddedaparagraphdescribingthegeneexpressionchangesinducedbyRBM10OE/KDin the result part and add one supplementary table listing all the genes differentially expressed upon RBM10perturbation.Notably, theexpression changes induced by KD and OE werenot inversely correlated,indicating these eventsmightnotbe directly regulated by RBM10.Seealsotheresponse to referee #2. Also does RBM10 bind to the mRNA UTRs, which are frequently involved in regulation of translation and RNA stability. The answers to these questions should already be in the PAR-CLIP and RNA-Seq data.Thefactthat the two patients presented in this study display milder phenotypes compared to the typical TARP syndrome, despite having impaired function of RBM10 in slicing wouldarguethattheproteinmayhaveadditionalfunctionsinthecytoplasm. RBM10bindsboth5’and 3’UTR (seeFigure2).However,comparing with coding sequences,the binding attheUTR regionsismuch lessenriched (seealso theresponseto referee#2.).Wetried to associate the UTR binding to the change of gene expression induced by RBM10 OE or KD, but could notobserve any clearcorrelation.Having said that,again we could notexclude the possible effect of UTR binding, such as translational control. We fully agree with the referee, given the milderphenotypepresentedinourpatients, it is conceivable that the mutantRBM10 might retain additionalfunctionsin the cytoplasm.To addressthisawaitsfuture study. Minorissues: 1.Figure1b.From themethodsitisunclearwhythevaluesfortheconsensusclustersarehigher than the values forthereads.Ifclustersareaggregatesofreads,onewould expectthecluster density to be less than the read density. Weapologizeforcausingsuchconfusion.IntheYaxis,itisthedensityofreadsandbinding clustersthatare represented,which are normalized by the totalno.ofreadsorclusters. 2.On Figure2ctheauthorsneed to definewhatisconsidered to be"Strong","Medium"and "Weak" binding site. Wedefinedintherevisedmethodpart.Seealsotheresponsetoreferee#2. 3.On Figure 3c showing the aligned PAR-CLIPreads,ratherthanatrianglewillbemore informative to the reader. In limited space,showing allthe aligned PAR-CLIPreadswouldmakethefigurequitemessy.Upon the acceptance of the manuscript, we will upload the PAR-CLIP dataset into the database server DORINA hosted in our institute (http://dorina/rbp_browser/dorina.html). In DORINA, all the EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 24 aligned readscould be displayed in an interactivemanner in UCSC genome browser.We believe this would be the best solution for visualizing our PAR-CLIPdata. Novelty Althoughassociationwith thespliceosomeraises thepossibility thatRBM10regulatesalternative splicing this has notbeen shown to date.Furthermore,the rich sequence data obtained in this study can provide significantinsightinto the mechanismsby which RBM10 regulatessplicing. Wethanktherefereeforhis/herappreciationofthenoveltyofourstudy. MedicalImpact Mutations in RBM10 have been associated with developmental disorders. Furthermore, it is frequently mutated in certain types of cancer. Understanding its function may contribute to developing cancertherapiesand prognosticmarkers. Wethanktherefereeforhis/herappreciationoftherelevanceofourstudy. Adequacyofthemodelsystem TheauthorsexpressepitopetaggedRBM10forthePAR-CLIPexperiments.Whilethisisacceptable, particularly in cases where good quality antibodies are not immediately available for the endogenous protein, the authors need to show that the levels of the expressed protein are comparable to those ofthe endogenousprotein.Maintaining physiological protein levels is critical asover-expression may resultin binding to low affinity siteson the RNA thatarenotoccupied under normalconditions. We are aware of the possible irrelevant effects induced by uncontrolled protein overexpression. Therefore,beforeperforming PAR-CLIPaswellasRNA-Seq,wemeasuredtheexpressionlevelof RBM10 under a series of concentration of doxycyline (DOX) and chose the one with the lowest induction level, i.e. approximately 2.5 fold overexpression comparing with endogenous expression level, induced by 10 ng/mL DOX (see Supp Fig. 2B) Referee#3(Remarks): RBM10isanRNA bindingproteinthathasbeenassociatedinseveralstudieswiththespliceosome. However, its function there has remained unclear. The work presented by Wang et al in this manuscriptascribes a function ofRBM10 in splicing and more specifically in exon recognition.The authors use a combination of PAR-CLIP and RNA-seq to build an integrated model for RNA splicing regulation by RBM10.Wang etalalso show thatalternative splicing patterns in patients with TARP syndrome resemble those of RBM10 knockout cell lines, concluding that the splicing regulatory function of RBM10 is disrupted in the patients. The results of the presented work can potentially have a significant impact on our understanding of splicing regulation in organism developmentand human disordersincluding cancer.Whilethequalityoftheraw data appearsto be adequatethesubsequentanalysisleavesa lotto bedesired (seethespecificcommentsfordetails). The results as presented in the manuscript do not support the proposed RBM10 binding site sequence and the modelfor splicing regulation by RBM10.There are also some issues thatneed to beaddressed in respectto themodelsystem.In particular,theuseofprotein over-expression in the PAR-CLIP experiments may result in the identification of binding sites that are not normally occupied by RBM10. If these deficiencies are adequately addressed the work by Wang et al will withoutdoubthavesignificantimpact. Wethanktherefereeforhis/herappreciationofthenoveltyandrelevanceofourstudy,and thank him/her for his/her critical comments. Following his/her suggestions, we carried out further computational and experimental work. We believe that our additional results substantially improved the quality of the paper and addressed therequestsmadeby thereferee. EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 25 2nd EditorialDecision 04 July 2013 ThankyouforthesubmissionofyourrevisedmanuscripttoEMBO MolecularMedicine. WehavenowheardbackfromthetwoReviewerswhomweaskedtore-evaluate yourrevised manuscript. YouwillseethattwooutofthreeReviewershaveremainingissuesthatpreventusfrom considering publication atthistime. Reviewer2notesthatthefindingthatRBM10bindstoU2snRNA opensuptodifferent interpretations of your data. S/he also challengestheconclusion thatRBM10 bindsGAAGA and wouldliketoseestrongerstatisticalsupportthatcorrectnucleotidecontrolswereconsidered.This RevieweralsonotesacertainambivalencewithrespecttotheresultsonexonicbindingofRBM10. Finally, Reviewer2 disagrees that the splicing changes from the patient correlate well with RBM10 knock down in 293T cellsand would likeyou to verify how specificthecorrelation isby checking otherlarge-scale datasets. Reviewer3hassomeremainingconcerns as welland is more bluntconcerning the data on the RBM10bindingsiteandtheCLIPdataandsuggeststhattheyberemovedaltogether,whilemoving supplementary figures S2 and 3 to the main body.Since Reviewer2 also has reservations on the CLIPdata Iwould suggestthatyou comply with Reviewer3'srequeston the CLIP data and include the RBM10 binding site data only if you can provide further experimental support as indicated by Reviewer2. Asyouknow,wewouldnormallynotallow asecondrevision.However,afterconsulting with an externalexpert,Iam prepared in thiscase,to give you anotheropportunity to improve your manuscript,withtheunderstandingthattheReviewers'concernsmustbefullyaddressedwith additionalexperimentaldata where appropriate.Please also provide an additionalcopy ofthe revised manuscriptwith the changes highlighted in colour. If you fully comply with the requested changes,the finaldecision willbe possibly made atthe Editoriallevel. I look forward to seeing a revised form of your manuscript as soon as possible. 2nd Revision 06 July 2013 I would like to thank you for giving us another opportunity to improve our manuscript.After reading carefully your suggestions and the remaining concerns raised by both referees, we would like to makethechangesasdescribedbelow. We feel that the complete removal of PAR-CLIP datasets, as recommened by referee 3, is not appropriate because 1) the strength of our study is that we identified not only splicing changes induced by RBM10 perturbation but also transcriptome-wideRBM10bindingsites.Onlyafterthe integrativeanalysisofboth datasetswecould revealthemolecularmechanismsunderlying RBM10 mediated splicing regulation. Removal of the whole PAR-CLIP result will make the manuscript incomprehsible. 2) Our PAR-CLIP result is solid. As shown in FigS1, we have extensively demonstrated the quality by different metrics as well as the biological replicates, which showed similar performance as other published PAR-CLIP datasets (e.g.Hafner M,Cell2010;Lebedeva S, Molecular Cell 2011). Especailly, the RBM10 intronic binding could be associated with RBM10 mediatedsplicingchangesandtheresultingmechanisticmodelcouldbeunambiguouslyvalidated withminigeneexperiments. As suggested by you and referee 2, we planned to take some unessential parts out or move into supporting information,which includes,EMBO MolecularMedicine PeerReview Process File - EMM-2013-02663 © EMBO 26 1)Weremoved Fig.1. 2) We removed ‘Sequence features associated with RBM10 binding’, although we think the enrichmentof GAAGA motifs close to exonic RBM10 binding sites could notbe explained by any nucleotide bias in exons, as claimed by referee 2. As described in Method section “Analysis of sequencefeaturesaround RBM10 binding sites”,wegenerated an appropriate background modelfor motifanalysisandcontrolsiteswerepickedonlyfrom exonswhendefiningexonicbindingmotifs. Wealsoavoidedanypossiblebiasduetoexonlengthordistancesbetweenbindingsitesandexon boundaries. 3) We moved the ‚RBM10 binds to U2 snRNA’ into Supporting information, as suggested by referee 2. 4) We feel the three minigene experiments are redundant and now left only the last one with RBM10-PUF fusionexperiment. 5)Weremoved thesecond mechanisticmodelwith theeffectofRBM10 exonicbinding,which has notbeen substantiated by independentvalidation experiments. Finally, as to the correlation between splicing changes from the patient and those induced by RBM10knockdown in 293T cells,itissignificant(p<10e-16)and given thatthey weremeasured in different cell types, a higher correlation coefficient should not be expected. Therefore we do not believe it is just some noise or stochastic effects and the comparison with the data from other splicing factorknockdown in 293 cells is notnecessary. I hope you would find the changes listed above acceptable.I am looking forward to your editorial decision.