Redirecting splicing with bifunctionaloligonucleotidesJean-Philippe Brosseau12 Jean-Francois Lucier13 Andree-Anne Lamarche3

Lulzim Shkreta3 Daniel Gendron1 Elvy Lapointe1 Philippe Thibault1 Eric Paquet1

Jean-Pierre Perreault12 Sherif Abou Elela13 and Benoit Chabot13

1Laboratory of Functional Genomics and Research Centre on RNA Biology of the Universite de SherbrookeSherbrooke Quebec J1E 4K8 Canada 2Department of Biochemistry Faculty of Medicine and Health SciencesUniversite de Sherbrooke Sherbrooke Quebec J1E 4K8 Canada and 3Department of Microbiology andInfectious Diseases Faculty of Medicine and Health Sciences Universite de Sherbrooke SherbrookeQuebec J1E 4K8 Canada

Received July 8 2013 Revised October 21 2013 Accepted November 18 2013

ABSTRACT

Ectopic modulators of alternative splicing areimportant tools to study the function of splicevariants and for correcting mis-splicing events thatcause human diseases Such modulators can bebifunctional oligonucleotides made of an antisenseportion that determines target specificity and a non-hybridizing tail that recruits proteins or RNAproteincomplexes that affect splice site selection (TOSS andTOES respectively for targeted oligonucleotidesilencer of splicing and targeted oligonucleotideenhancer of splicing) The use of TOSS and TOEShas been restricted to a handful of targets To gen-eralize the applicability and demonstrate the robust-ness of TOSS we have tested this approach on morethan 50 alternative splicing events Moreover wehave developed an algorithm that can design activeTOSS with a success rate of 80 To producebifunctional oligonucleotides capable of stimulatingsplicing we built on the observation that bindingsites for TDP-43 can stimulate splicing and improveU1 snRNP binding when inserted downstream from50 splice sites A TOES designed to recruit TDP-43improved exon 7 inclusion in SMN2 Overall ourstudy shows that bifunctional oligonucleotides canredirect splicing on a variety of genes justifying theirinclusion in the molecular arsenal that aims to alterthe production of splice variants

INTRODUCTION

Alternative splicing is the process by which exons aredifferentially combined to produce different types of

mRNAs from a single gene This process allows cells toproduce an average of 8ndash10 splice variants per gene sub-stantially increasing the coding capacity of the humangenome (1ndash3) and making an immense contribution tothe structural and functional diversity of our proteome(4) Defects in alternative splicing contribute to manyhuman diseases including spinal muscular atrophy(SMA) myotonic dystrophy and cancer (56) Inaddition it is estimated that as many as 60 of humandiseases might be caused by point mutations that altersplicing (eg b-thalassemia cystic fibrosis and progeria)(7) However the function of the majority of splicingisoforms and the real number of human diseases affectedby splicing remain unclearMolecular tools that can specifically alter the proportion

of splice variants are essential to assess the function of amultitude of splice variants Unfortunately deducing thefunction of splice variants by RNA interference appro-aches is challenging because decreasing the level of agiven splice variant also changes the total amount ofproducts for that gene (8) Therefore alternativeapproaches are needed to alter the production of splice vari-ants by redirecting splicing decisions without changingthe overall level of gene expression The original strategypioneered by the group of Kole used an antisense oligo-nucleotide (ASO) complementary to a cryptic splice sitein the b-globin gene that prevented its use and favoredselection of the authentic site (9) This approach has sincebeen used regularly to alter the proportion of splicevariants produced from mutated genes or alternativesplicing units [(10) reviewed in (1112)] Since a shift insplicing does not in principle alter the absolute amount ofgene products this approach increases the confidence ofattributing a function to a specific splice variantGiven that alternative splicing decisions are often

controlled by regulatory proteins bound to exonic and

To whom correspondence should be addressed Tel +819 820 6868 (ext 75321) Fax +819 820 6831 Email benoitchabotusherbrookeca

Published online 26 December 2013 Nucleic Acids Research 2014 Vol 42 No 6 e40doi101093nargkt1287

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc30) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited For commercialre-use please contact journalspermissionsoupcom

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intronic elements located in the vicinity of alternativesplice sites the ASO approach has evolved to targetthese elements and prevent them from recruiting regula-tory proteins (13ndash15) This new strategy was used success-fully to abrogate the action of an intronic splicing silencerwithin the SMN2 gene increase exon 7 inclusion andimprove the SMA-associated cellular phenotype (16ndash18)Splice switching oligonucleotides in the same designcategory are being tested for other diseases [reviewedin (1219)] including Duchenne muscular dystrophy(20ndash22)Another splice switching strategy is to use oligonucleo-

tides that contain a portion complementary to the targetsite linked to a non-hybridizing tail that can provide ei-ther stimulatory or repressor function When the tailcontains binding sites for hnRNP A1 positioning suchan oligonucleotide upstream of a 50 splice site (50ss) inter-feres with U1 snRNP binding and repress splice site use(23) This bifunctional oligonucleotide design has beencoined TOSS for targeted oligonucleotide silencer ofsplicing (11) Although the inhibitory potential of tailsbound by other proteins has not been examined systemat-ically exon-binding oligonucleotides with tails carryingsplicing signals also displayed strong inhibitory activity(2324) TOSS with A1 tails have been used successfullyto repress exon 8 in SMN2 hence redirecting splicing tofavor exon 7 inclusion in the fibroblasts of SMA patientand in a mouse model of SMA (25) In contrastbifunctional A1 binding oligonucleotides positioned inintrons can stimulate splicing of long introns in vivo andcan elicit skipping of an intervening 50ss in splicingextracts (26)Bifunctional oligonucleotides carrying a tail designed

to stimulate splice site usage are coined TOES fortargeted oligonucleotide enhancer of splicing (27) Thiscategory includes oligonucleotides that contain a tailthat recruits positively acting SR proteins (28) or a tailmade of a synthetic RS domain covalently linked to anantisense moiety (29) A splicing enhancer element wasalso engineered in the U7 snRNA sequence which whenexpressed in SMA cells stably stimulated SMN2 exon 7inclusion (30)To assess the biological function of a growing reper-

toire of splice variants we need approaches that canchange the relative abundance of such variantsAlthough bifunctional oligonucleotides are in principleideally suited to assess isoform function their use hasremained restricted to only a handful of cases Tovalidate the broad applicability of bifunctional oligo-nucleotides we show that TOSS can repress splice siteuse on a wide spectrum of targets leading to an algo-rithm that can design active TOSS with a success rate of80 As for the alternative strategy that aims to stimu-late splice site utilization we describe a new TOES designthat uses a TDP-43 binding tail to promote exoninclusion Our results validate the use of bifunctionaloligonucleotides to alter splicing decisions on an expand-ing repertoire of targets and make them attractiveas individual or high-throughput tools to modulate theproduction of splice variants

MATERIALS AND METHODS

Cell culture and transfection

SKOV3ip1 NIH-OVCAR-3 PC-3 ZR-75-1 and OVC-116 cell lines have been described previously (31) TOSSand ASO were synthesized as 20OMe and standardlydesalted by IDT (USA) Oligonucleotides purity wasassessed by fractionation on 15 denaturing acrylamidegels Oligonucleotides were diluted in Opti-MEM to whichan equivalent volume of Lipofectamine 2000 was addedThe mixture was added to cells that had been previouslyseeded in a 6- or 96-well plate with complete media Thefinal concentration of oligonucleotide was 150 nM forTOES and 400 nM for TOSS and ASO TOSS and ASOwere transfected in biological triplicates (three transfec-tions using different cell passages)

RNA extraction and RT-PCR

RNA was extracted 24 h post-transfection using eitherTrizol (Invitrogen) or a silica-based column (AbsolutelyRNA 96 Microprep kit from Stratagene) (32) Wefollowed the manufacturerrsquos instruction for Trizol(Invitrogen) extraction except that linear acrylamide(5 mg) was added during isopropanol precipitationIntegrity and quality of RNA was evaluated by AgilentBioanalyzer and Nanodrop respectively The level ofcontaminating genomic DNA was examined as describedelsewhere (33) The TOSS-induced splicing shift wasevaluated by endpoint RT-PCR and quantitative RT-PCR The design of endpoint RT-PCR primers was per-formed as described previously (33) The endpoint PCRassays were performed using the Qiagen One step RT-PCR kit (Qiagen) using the gene-specific reverse primerin the reverse transcription step PCR products werefractionated on a Caliper 90 workstation as describedpreviously (33) The percent of splicing index (Psi or cvalue) were calculated for each sample and the c(cLFcTOSS) was used to monitor the efficiency ofTOSS-induced splicing shift Design and validation ofquantitative RT-PCR assays were done as previouslydescribed (3234) For each alternative splicing eventsfollowed by endpoint RT-PCR a long-specific a short-specific and a global (targeting all isoforms) primer pairswere designed whenever possible Following a randompriming strategy (random hexamers) and quantitativeRT-PCR fluorescence measurement using SYBR Greentwo analytical strategies were used One is the relative ex-pression normalized by housekeeping as previouslydescribed (32) The second is the quantitative splicingindex which corresponds to the percentage of the longerisoform over the sum of the short and long isoforms (32)

Western analysis

Proteins were separated by SDSndashPAGE and transferredonto nitrocellulose Hybond C (Amersham) using atransfer buffer (145 glycine 03 Tris-base and 25methanol) for 6 h at 70V and 4C The membrane waswashed with TBS (024 TrisndashHCl 08 NaCl pH80) for 5min and then incubated for 15 h at 4C withTBST (TBS+005 Tween 20 and 5 milk powder)

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After blocking the membrane was incubated with theantibody at room temperature for 2 h The membranewas washed twice with TBST and the secondaryantibody was added and incubated for 1 h Detectionwas performed with ECL (Amersham Biosciences) accord-ing to the manufacturerrsquos recommendations

In vitro splicing assay and oligonucleotide-mediatedRNase H protection assay

Splicing assays were performed as described (35) usingHeLa nuclear extracts (36) RNase H protection assaywas performed as follows to 4 ml of HeLa extract areadded 05 ml of rATP 125mM 05 ml of MgCl2 80mM05 ml of creatine phosphate 05M 25ml of PVA 13025 ml of DTT 01M 025 ml of RNAguard 40Uml025 ml of KCl 1M 1375 ml of buffer D (35) and 025 mlof RNase H 5unitsml One microliter of radiolabeledRNA is added (30 000 cpm) and 1ml of creatine kinase(1Uml) Mixtures were incubated 0 5 or 20min at 30Cand then placed on ice In total 075 pmol of oligonucleo-tides 7A-A5 and 7B-A5 (35) are added with 25 U ofRNase H and incubation is carried out for 15min at30C RNA is then phenol extracted after adding 500 mlof extraction buffer (03M sodium acetate 02 of SDS)and ethanol precipitated RNA is resuspended informamide dyes boiled loaded and fractionated on adenaturing acrylamide gel

Construction of the inducible shTDPHeLa cell line

Oligonucleotides corresponding to TDP-43 were shTDP-43Bfwd 50-GATCCCACTACAATTGATATCAAATTCAAGAGATTTGATATCAATTGTAGTGTTTTTGGAAA-30 and shTDP-43Brev 50-AGCTTTTCCAAAAACACTACAATTGATATCAAATCTCTTGAATTTGATATCAATTGTAGTGGG-30 After duplex formation theywere inserted at the BglII and HindIII sites in plasmidpTER + which was transfected in HeLa cells alreadyexpressing the transcriptional repressor (via expressionof plasmid pCDNA 6TR) Four hours post-transfectionDMEM with 400mgml of zeocine and 3 mgml ofblasticidine was added to select for the presence ofboth plasmids Distinct clones were isolated and inducedwith 15 mgml of doxycycline to test for the depletion ofTDP-43

RESULTS

Measuring TOSS efficacy

We have shown previously that a bifunctional interferingoligonucleotide (TOSS A1_Bclx4) containing a 20OMe tailwith hnRNP A1 binding sites and a sequence complemen-tary to the 4 to 24 region upstream of the Bcl-xL 50ssregion can shift Bcl-x splicing from the long variant (Bcl-xL) to the short variant (Bcl-xS) in human PC-3 andHCT-116 cells (23) (Figure 1A and B) Quantitative RT-PCR indicates that the shift in the production of Bcl-xvariants is caused by a reduction in Bcl-xL and anincrease in Bcl-xS (Figure 1C) suggesting that the repres-sive effect of the TOSS on the 50ss of Bcl-xL improves thecompetitivity of the 50ss of Bcl-xS However it is difficult

to exclude the possibility that the observed effect was atleast in part due to the interference of the TOSS with thereverse transcription step required for PCR analysis(Supplementary Figure S1a) To evaluate this possibilitywe measured the effects of adding TOSS to total RNAprior to reverse transcription and PCR amplificationIncreasing amounts of TOSS targeting three differentgenes (Bcl-x APAF1 and CAPN3) were added immedi-ately before the reverse transcription step and the splicingpattern determined using PCR In all cases the synthesisof the longer product was inhibited even at concentra-tions as low as 4 nM (Supplementary Figure S1b) Thisresult suggests that the TOSS remaining in the totalRNA samples extracted from transfected cells may alterthe splicing isoform ratio and lead to an overestimation ofTOSS efficiency The impact of TOSS on the splicingpattern estimated by PCR varied based on the procedureused for RNA extraction Column-based purification pro-cedure did not fully remove the TOSS in RNA samples(37) while Trizol-based extraction removed the oligo-nucleotide from the aqueous phase (SupplementaryFigure S2) and eliminated the artifactual impact ofTOSS during RT-PCR (Supplementary Figure S3)However while Trizol extraction eliminates the RT-PCRartifact this step is inconvenient when multiple samplesmust be analyzed in a high-throughput manner Sincenone of the commercially available high-throughputRNA purification kits tested removed TOSS from theRNA samples modifications of the RNA extraction pro-cedure do not provide a practical approach for the elim-ination of TOSS interference when detecting alternativesplicing using RT-PCRTo obtain an unbiased estimate of the in vivo impact of

TOSS on the production of splice isoforms we performedquantitative RT-PCR to detect the short and long variantsindividually While the long variant values could vary de-pending on the method of RNA purification the valuesfor the short variant were similar as expected becausethese variants lack sequences complementary to theTOSS (Supplementary Figure S4) Measuring the shortvariant by quantitative RT-PCR therefore accuratelyassess the shift in splicing regardless of the presence ofTOSS in the reverse transcription reaction We concludethat if Trizol extraction cannot be accommodated thereliable monitoring of the effects of bifunctional oligo-nucleotides requires PCR methods that exclude the amp-lification of the targeted sequenceTo further confirm the PCR estimates of the TOSS-de-

pendent splicing shift we analyzed the ratio of the proteinisoforms after exposure to TOSS and compared it to thePCR pattern As shown in Figure 1D the western blot ofCASP8 and CASP10 isoforms which are used as modelgenes were altered in the same direction and produced asplicing ratio similar to that detected by PCR using thesame samples (37)

Structural elements affecting TOSS efficacy

With the goal of improving the design of TOSS weevaluated some of the parameters that affect TOSSactivity To control intra- and intermolecular secondary

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structures that could interfere with the interaction ofTOSS with its target RNA we use the UNAFoldsoftware package (38) to predict the minimal G ofTOSS By examining the impact of previously testedTOSS with different G we found that a G superiorto 94 kcalmol which is two standard deviations awayfrom the average free energy of a thousand randomlydesigned TOSS generally yielded TOSS with efficientand reproducible modulation of alternative splicingOther parameters were taken into account such as GCcontent polynucleotide repeats and innate immuneresponse activating sequences (Figure 2)We also evaluated the general requirement of the tail of

TOSS because although it was previously shown that a tailthat binds hnRNP A1 was required for maximal modula-tion of Bcl-x splicing it was not clear if this was true for

other target genes (23) Therefore we generated ASOscontaining (TOSS) or lacking a tail (ASO) against 10 dif-ferent splicing events and monitored their impact onsplicing A splicing shift was acknowledged when eitherthe short or long splice variant changed by at least 2-fold As shown in Figure 3 eight ASOs modulatedsplicing without affecting the overall level of gene expres-sion while the ASO targeting SYK affected both splicingand global gene expression On the other hand seven ofthe eight TOSS that modulated alternative splicing didso in a manner superior to the corresponding ASO(Figure 3) We also noted a large increase in shortisoforms when TOSS were targeting CCNE1 KITGLIG4 and MCL1 ruling out any over estimation of thesplicing shift by the interfering RT-PCR artifact Even ifresidual amounts were present we observed that ASO

A

A1

505 189143 16295 5 5

Bclx - TOSS 1

236

B Endpoint PCR

505 189236 1629

505236 1629

Alternative 5

Quantitative PCR

Global

Short-specific

Long-specific

D

Short isoform

Long isoform

TOS

S 1 Ctrl -Mock

CASP8

TOS

S 1 Ctrl -Mock

Short isoform

Long isoformCASP10

C

30

20

10

00

Bclx - T

OSS 1

Ctrl - T

OSSMoc

k

rela

tive

expr

essi

on

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GlobalShortLong

0

60

40

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Figure 1 Impact of TOSS on the production of apoptotic splice variants (A) Schematic representation of the Bcl-x splicing unit and the location ofBclxndashTOSS1 (B) Positions of the PCR primers used to quantify splice variants of Bcl-x (C) Histograms representing average percentage valuesobtained by endpoint RT-PCR (upper panel) or the relative expression of variants by quantitative RT-PCR (bottom panel) in three biologicalreplicates PC-3 cells were transfected with BclxndashTOSS1 (400 nM final concentration) or a control TOSS carrying an antisense sequence not com-plementary to Bcl-x (ctrlndashTOSS) Total RNA was extracted with Trizol 24 h later (D) Western analysis of TOSS-induced shifts in CASP proteinisoforms HeLa cells were transfected with CASP8ndashTOSS1 and a mutated version (ctrlndashTOSS) containing four mismatches (upper panel) orCASP10ndashTOSS1 and a mutated version (ctrlndashTOSS) containing two mismatches (bottom panel) Proteins were extracted 72 h later and the pro-portion of the splice variants was verified by western analysis

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Figure 2 Development of the algorithm for designing TOSS An application programming interface (API) and web application was developed usingPerl version 588 (the Perl directory httpwwwperlorg) with CGIApplication HTMLTemplate and Bioperl CPAN modules (CPANComprehensive Perl Archive Network httpcpanorg) (A) To perform TOSS prediction the user specifies the alternative exon sequenceThe algorithm then extracts all possible sequences from the target sequence using a window of 20 nucleotides Each 20mer is evaluated for validGC content (between 30 and 80) the absence of four consecutive identical nucleotides and of immune stimulatory responses sequence motifs(39ndash42) and self-complementarity of the sequence (G higher than 94 kcalmol) Self-complementarity minimum energy folding was calculatedusing the hybrid-ss-min software included in the UNAFold package (38) using default parameters The G threshold was determined by calculatingthe average and standard deviation of 10 000 randomly generated 20mer sequences located at the 50 end of TOSS The threshold was fixed as theaverage plus two standard deviations to ensure that no strong intramolecular folding structure (lower than 94 kcalmol) was formed(B) Predictions that meet the above criteriarsquos are then sorted according to different filters First each prediction is submitted to a Blast analysis(43) against an in silico generated transcriptome database derived from the Aceview annotation (44) This step is performed to determine potential

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(continued)

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interfered with RT-PCR in a manner quantitativelysimilar to TOSS (Supplementary Figure S5) Thus thegreater shifting activity of TOSS in cells cannot be ex-plained by an artifactual impact on RT-PCR Moreoverthis increased efficiency in splicing modulation can directlybe attributed to the recruitment of hnRNP A1 to the tailbecause mutations in the tail reduced the effect on splicingin all cases (Supplementary Table S1 SupplementaryFigure S6)In comparison to RNA or DNA the 20OMe compos-

ition of the tail remains the most efficient to promote exonskipping on the KITLG pre-mRNA (SupplementaryFigure S7) as shown previously for modulation of 50ssselection on Bcl-x (23) Although tails carrying abranched structure a branch site or a 50ss can providerepressor activity (2324) we have used the hnRNP A1tail for the remainder of the current TOSS analysis

Parameters of targets for optimal TOSS modulation

TOSS efficiency may be affected by the distance that sep-arates its hybridization site from the target splice siteIndeed in vitro studies on Bcl-x suggested that theimpact of a TOSS decreased as the distance between itsbinding site and the targeted 50ss increased (23) To deter-mine if this observation applies to other pre-mRNAsin vivo we tested TOSS harboring sequences complemen-tary to various positions upstream of a 50ss in two genes(MCL1 and SHMT1) The results indicate that the distalpositioning of TOSS (31 and 38 on MCL1 and 49and 58 on SHMT1) was the most efficient and that adownstream exonic site either reduced TOSS efficiency orwas as active (Figure 4) A TOSS targeting an intronicregion downstream of an alternative 50ss in KITLG hadno impact (Supplementary Table S1) Although we havenot identified the upstream limit for the impact of TOSSefficient TOSS activity has so far been obtained by target-ing the 1 to 60 region from the target 50ss The positionthat imposes maximal activity likely varies between targetsmost probably due to differences in secondary structurethe presence of regulatory elements and the existence ofoff-target hybridization sites that could reduce the effect-ive concentration of TOSSTo increase the utility of the different parameters

influencing the modulation of alternative splicing webuilt a web interface-based program for TOSS designthat takes into account structural and target featuresbased from the above and our previous studies(232432) (Figure 2 httptosslgfusca) We use theprogram to design TOSS targeting more complexpre-mRNAs NUP98 carries three competing 50ss

(Figure 5A) and we used two TOSS complementary to aregion upstream of the more downstream 50ss (Figure 5A)Using endpoint RT-PCR (Figure 5B) we monitored thelevel of inclusion of the long variant (Figure 5C) NUP98ndashTOSS1 had little impact whereas NUP98ndashTOSS2decreased the production of the long variant more import-antly than NUP98ndashASO2 (Figure 5C) Quantitative RT-PCR confirmed that the level of the short splice variantwas strongly and specifically increased by NUP98ndashTOSS2(Figure 5D)

To determine whether a TOSS could affect the splicingof an exon flanked by adjacent alternative exons wetargeted three double cassette exons units (APP FGFR2and FANCA) and three units with triple adjacent alterna-tive exons (PTPN13 NRG1 and BCAS1) For each eventa TOSS was designed to target the first or the last alter-native exon Quantitative and endpoint RT-PCR wereused to evaluate exon-specific and multiple exon splicingExon-specific modulation was noted in five of six cases(APP FGFR2 FANCA PTPN13 and BCAS1Supplementary Figure S8ndashS13) and a reduction inglobal expression was also observed in one case(BCAS1 Supplementary Figure S13) Overall the TOSSremained specific to the targeted exon as the impact ofTOSS on the splicing of non-targeted adjacent alternativeexon was never observed

In conclusion we have tested a total of 89 TOSS andcontrol oligonucleotides The lsquowinnerrsquo designs proposedby the TOSS algorithm were active in 80 of the cases(24 of 30) as judged by endpoint RT-PCR(Supplementary Table S1) When Trizol extraction wasnot used for purifying RNA quantitative RT-PCR con-firmed that 17 of the 20 TOSS-mediated shifts were notartifacts caused by the presence of TOSS in the endpointPCR mixtures Although we have focused our studyon the ability of TOSS to target 50ss modulating alterna-tive 30ss usage was attempted once with success(Supplementary Figure S14) Notably some ASO andTOSS promoted global reduction in expression but thereasons for these effects are unclear Thus our study dem-onstrates that TOSS provides a robust approach that canpromote dramatic shifts in the ratio of splice variants withlimited impact on global expression levels

Development of a TOES that promotes exon inclusion

The antisense technology currently applied to splicingcontrol mostly aims at preventing the binding of splicingfactors However in some instances it may be useful toimprove splice site recognition One technology that hasbeen developed to stimulate exon inclusion has been

Figure 2 Continuedoff-targets associated with the hybridizing portion of the TOSS Second the sequence complementary to the hybridizing portion of the TOSS isevaluated for the potential presence of splicing enhancer (ESE) and splicing silencer (ESS) motif sequences To perform this step each nucleotidefound in the input sequence was scored against a database of potential ESE and ESS motifs previously identified by Stadler et al (45) If thenucleotide is part of an ESE or an ESS motif the nucleotide score is increased or decreased by 1 respectively The TOSS prediction is then attributeda global ESEESS score representing the sum of the nucleotides Then valid predictions are separated in two pools based on 50ss distances the firstand second pool being respectively below and above a distance of 30 nucleotides from the 50 splice junction Once all filters are computed bothpools are then ordered to favor predictions with the least potential off-targets and the highest global ESEESS score to maximize disruption ofpotential enhancers Finally predictions from the pool closest to the 50ss (lt30 nucleotides) are favored over the long distance pool The program webapplication can be accessed at httptosslgfusca

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Figure 3 Effect of TOSS versus ASO (A) Schematic representation of a standard cassette exon The positions of qRT-PCR primers are indicated(BndashI) SKOV3ip1 cells were transfected with specific TOSS their ASO versions (lacking a tail) and a control TOSS carrying no complementarity tothe targeted gene Total RNA was extracted 24 h later using Absolutely RNA 96 Microprep kit (Stratagene) Histograms represent average relativevalues for global expression (dark gray) short isoform (white) and the long isoform (black) obtained by quantitative RT-PCR in three biologicalreplicates (upper portion of each panel) The quantitative RT-PCR values for the short and long isoforms were output as percentage of long variantfor each condition (lower portion of each panel) using calculations described previously (32)

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bifunctional oligonucleotides that can recruit SR proteins(28) We tested a TOES design that uses a phospho-rothioate SRSF1 binding tail (28) Alternative unitsin LGALS9 and KITLG were targeted with SRSF1-recruiting TOES and splicing was monitored usingPCR As expected the TOES promoted exon inclusionin both cases (Supplementary Figure S15) However wefound that the phosphorothioate tail required for TOESactivity was toxic for our ovarian and breast cancer celllines which makes these TOES difficult to use in func-tional assays To avoid the toxic effects we replaced thephosphorothioate tail with a 20OMe tail which hasworked well for splicing repression using hnRNP A1Unfortunately TOES with 20OMe tails failed to stimulatesplicing presumably due to the lack of binding ofSRSF1 (Supplementary Figure S15) We conclude that

phosphorothioate TOES or 20OMe TOES using SRSF1are not practical for analyzing the impact of alternativesplicing on cell functions

To design TOES capable of modulating splicingwithout affecting cell viability we focused on TDP-43This RNA-binding protein and splicing factor can bindwith similar affinity to single-stranded TG and UGrepeats (46) suggesting that its binding may tolerate20OMe nucleotide modifications Its relatively high abun-dance also decreases the possibility that its partial seques-tration by nanomolar quantities of TOES will impactnormal TDP-43-mediated functions The enhancingcapacity of TDP-43 was tested by inserting the TDP-43binding element (TBS) (UG)13 downstream of a 50ss in themodel Dup 51 minigene which contains duplicatedb-globin exon and intron sequences (Figure 6A) Since

Figure 4 Impact of the distance relative to 50ss on TOSS activity (A) Schematic representation of MCL1 (left panel) and SHMT1 (right panel)cassette exon and the relative positions of TOSS to 50ss (B) SKOV3ip1 cells were transfected with TOSS harboring sequences complementary tovarious positions upstream of a 50ss of the two genes (MCL1 and SHMT1) and a control TOSS carrying no complementarity to the targeted genes(MCL1_TOSS_2 position 38 MCL1_TOSS_3 position 31 MCL1_TOSS_1 position 2 SHMT1_TOSS_2 position 58 SHMT1_TOSS_3position 49 and SHMT1_TOSS_1 position 0) Total RNA was extracted 24 h later using Absolutely RNA 96 Microprep kit (Stratagene)Histograms represent average relative values for global expression (dark gray) short isoform (white) and the long isoform (black) obtained byquantitative RT-PCR in three biological replicates (upper panel) (C) Histograms represent average percentage values obtained by end-point PCR inthree biological replicates

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skipping of the central exon was stimulated the TBSappeared to improve selection of the 50ss of the firstexon relative to that of the central alternative exon(Figure 6B) This splicing shift was abrogated when cellswere depleted of TDP-43 through expression of a shRNA(Figure 6B and C) It is possible that exon skipping in Dup51 is caused by repression of the internal exon To addressthe mechanism by which TDP-43 controls splice site selec-tion we tested the impact of inserting UG repeats in modelpre-mRNAs carrying a single intron In the two differentmodel pre-mRNAs tested the presence of UG repeatspositioned downstream from the 50ss stimulated splicingin HeLa nuclear extracts (Supplementary Figure S16)indicating that intronic TDP-43 binding sites stimulatethe use of the upstream 50ss

Next we tested the TBS in a different model pre-mRNA(553) carrying two competing 50ss (Figure 7A) as a preludeto measuring the impact of the TBS on U1 snRNPbinding Pre-mRNA splicing in a HeLa nuclear extractindicated that the TBS shifted splicing to favor the 50ssdirectly upstream from the TBS (Figure 7B) We used thispre-mRNA to analyze U1 snRNP binding to thecompeting 50ss through an oligonucleotideRNAse H pro-tection assay (35) and observed that the TBS improved U1snRNP binding to the 50ss directly upstream from it(Figure 7C and D)

Finally we designed (UG)10-carrying TOES targetingthe disease-related human SMN2 gene (Figure 8A)Improving the inclusion of alternative exon 7 in SMN2is used as a strategy to increase the production of theSMN protein in SMA patients who have lost the SMN1gene (131419) The antisense portion of the TOES wascomplementary to positions +21 to +35 downstream ofthe 50ss of exon 7 a position previously shown to be inef-fective when targeted by an ASO (14) TOES were trans-fected in the Sma77 cell line derived from a SMA patientAs shown in Figure 8B inclusion of exon 7 remained un-changed when the complementary ASO was used (lane 2)consistent with previous observations (14) In contrast thepresence of the (UG)10 tail at the 5

0 end of the TOES oligoincreased exon inclusion (lane 3) a larger increase wasobtained by positioning the TDP-43 binding tail at the30 end of TOES possibly because of closer proximity ofthe tail to the target splice site (lane 4) As expected thehigher level of the exon 7-containing SMN2 splice variantimproved production of the SMN2 protein (Figure 8C)Because SMA cells have limited growth potential and donot support well successive transfections we have beenunable to confirm that the splicing shift in SMA cells iscaused by TDP-43 Thus while we would argue that theactivity of the trans-acting TBS is mediated by TDP-43 (aswas the cis-acting TBS in HeLa cells) we cannot rule out

Figure 5 Using TOSS to redirect splicing in a complex unit carrying alternative 50ss (A) Schematic representation of the NUP98 alternative splicingunit as well as the position of NUP98ndashTOSS1 and NUP98ndashTOSS2 on the 222 nt exon (B) Positions of primers used to amplify splice variants ofNUP98 in endpoint and quantitative PCR assays (C) SKOV3ip1 cells were transfected with NUP98ndashTOSS1 NUP98ndashTOSS2 their ASO versions(lacking a tail) and a control TOSS and ASO carrying no complementarity to NUP98 (ctrlndashTOSS and ctrlndashASO respectively) Total RNA wasextracted with Trizol 24 h later Histograms representing average percentage values of the long variant obtained by endpoint PCR in three biologicalreplicates are depicted (D) SKOV3ip1 cells were transfected with NUP98ndashTOSS1 NUP98ndashTOSS2 and a control TOSS carrying an antisensesequence not complementary to NUP98 (ctrlndashTOSS) Total RNA was extracted 24 h later using the Absolutely RNA 96 Microprep kit(Stratagene) Histograms represent average relative values for global expression (dark gray) and the short (white) splice variant obtained by quan-titative RT-PCR in three biological replicates

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that other UG repeats binding proteins are contributing tothe shift in SMA cells The TOES containing a TDP-43binding site therefore represents an interesting toolfor improving the relative inclusion of SMN2 exon 7We conclude that TOES with TDP-43 binding sites area practical alternative for improving exon inclusion andfor monitoring the impact of splicing shifts on cell func-tions since no apparent toxicity was noted followingtransfection

DISCUSSION

Alternative splicing is a powerful generator of protein di-versity and profiles of alternative splicing are often alteredin human diseases from muscular dystrophy to cancerProcedures that can specifically decrease the level ofsplice variants or redirect splicing to favor the productionof others can be useful to address the function of the con-tinuously expanding repertoire of splice variants and tocorrect or interfere with the production of splice isoformsin human maladies

TOSS

To generalize the use of bifunctional oligonucleotides astools to modulate the production of splice variants we

have developed an algorithm that can design functionalTOSS with a success rate of 80 Using this program wehave successfully targeted 50ss in genes displaying a varietyof architecture including competing 50ss as well as cassetteexons in simple or more complex splicing units By target-ing a region upstream of an alternative 50ss there might beadditional impact on gene expression since the hybridizedTOSS or ASO may interfere with the deposition of theexon junction complex possibly compromising mRNAtransport and stability Further interference may occurat the translational level In all cases however alteringthese processes would contribute to the ultimate goalwhich is to repress specifically the expression of thesplice variant While most of the splicing shifts hadminimal impact on the overall expression levels someASO and TOSS promoted decreases in global expressionThe reasons for these effects are not understood but onepossible mechanism may be chromatin-mediated asshown for the Ago-mediated action of siRNAs (48)

Although we have not systematically investigated theability of TOSS to modulate 30ss choice our singleattempt at it was encouraging suggesting that it shouldbe possible to extract rules to achieve maximal interfer-ence on splicing units with different 30ss configurationsAll the TOSS that display repressing activity were

Figure 6 Impact of TDP-43 binding sites on splicing (A) The Dup 51 minigene was used as model (47) The () is the wild-type version thatcontains an alternative cassette exon while the (+) version contains a (UG)13 element (TBS) in the first intron 90 nt downstream from the 50ss and47 nt upstream of the 30ss of the internal exon (B) In vivo splicing of () and (+) in induced (I shTDP-43) and non-induced (NI) HeLashTDP-43 cells Following an induction or mock-induction period of 48 h with doxycycline plasmids were transfected and RNA collected 24 h laterRT-PCR products were fractionated on denaturing acrylamide gels and the relative level of exon inclusion was calculated from biological triplicates(C) Western analysis of TDP-43 expression in the induced (I shTDP-43) and NI HeLashTDP-43 cell line following doxycycline induction for thetimes indicated

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complementary to the exonic region upstream of thetargeted 50ss (up to position 60 was tested) The tailportion of the TOSS was important in the majority ofthe cases since a tail-less ASO was less likely to work orits impact was consistently lower than that of a TOSSAlthough we have limited our current study to a tailbound by the hnRNP A1 protein we have shownthat tails of various architectures can repress splicing(24) suggesting that it should be possible to design re-pressing tails that recruit other RNA-binding proteinsprovided that binding is not disrupted by the chemicalmodification used to improve the stability or delivery ofthe oligonucleotide

Interestingly bifunctional oligonucleotides with anhnRNP A1-bound tail can also be used to stimulatesplicing when they are positioned downstream of a 50ss(49) This activity has been associated with the presenceof additional hnRNP A1 binding sites located furtherdownstream in the intron and we have proposed thatstimulation occurs when an interaction between multiplebound hnRNP A1 approximate the ends of the intron tofavor spliceosome assembly (49) A similar A1A1 inter-action across an exon may lead to repression (5051)possibly explaining why intronic A1 binding elements canalso be silencers (18) On the other hand we have observedthat recruiting hnRNP A1 downstream of the 50ss in

Figure 7 TDP-43 stimulates U1 snRNP binding (A) Structure of the model pre-mRNAs used to determine the in vitro impact of a binding site forTDP-43 [TBS made up of (UG)13] on 50ss selection The 553 () pre-mRNA carries two competing 50ss (distal and proximal) The 553 (+) pre-mRNA derivative carries a (UG)13 TBS element 30 nt downstream of the distal 50ss (B) In vitro splicing of the two model pre-mRNAs for 90min ina HeLa nuclear extract The percentage of skipping of the proximal 50ss is indicated (C) Uniformly radiolabeled transcripts are incubated 0 5 or20min at 30C in a HeLa nuclear extract Two DNA oligonucleotides complementary to each 50ss are added along with RNase H which will cut theRNA moiety of the DNARNA duplex Transcripts are therefore cut when U1 is not bound to the 50ss Cleavage products are separated on adenaturing acrylamide gel The bands corresponding to double protection proximal 50ss protection only or distal 50ss protection only are quantitatedon PhosphorImager The table indicates the normalized percentage of U1 snRNP occupancy at both 50ss at only the proximal 50ss or only the distal50ss after incubation for 0 5 and 20min in a HeLa nuclear extract at 30C

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KITLG had no effect Thus the impact of an hnRNP A1binding site located downstream of a 50ss may vary accord-ing to the presence and position of additional high affinitysites for hnRNP A1 on the pre-mRNA

TOES

Considerably less attention has been devoted to oligo-nucleotides designed to stimulate splicing by recruitingor mimicking splicing regulators Furthermore we foundthat phosphorothioate oligonucleotides were cytotoxicand that 20OMe versions designed to recruit the positivesplicing factor SRSF1 were inactive most likely becausethey were incompatible with efficient protein bindingPossibly for the same reason we have been unable todocument the activity of 20OMe tails with sequences

normally recognized by RBFOX proteins (data notshown)

Because the above observations limited our ability totest TOES on a variety of targets we instead aimed atidentifying a protein that could stimulate 50ss usagewhen positioned downstream from a 50ss and that couldact when recruited by a tailed 20OMe oligonucleotide Wefocused on TDP-43 because this hnRNP-like proteindisplays similarly strong binding to RNA and DNA(46) suggesting that it could also interact with a 20OMe-modified sequence While a role for TDP-43 in 30ss controlis well established (46) a function in 50ss selection is moreambiguous (52) We first showed that cis-acting TDP-43binding sites positioned downstream of a 50ss could stimu-late splicing in two different model genes in one case the

Figure 8 TOES-mediated exon inclusion on SMN2 transcripts (A) A portion of the SMN2 gene was targeted with TDP-43-TOES complementaryto positions+21 to+35 downstream of exon 7 The portion of the oligos complementary to SMN2 is underlined (B) RNA was extracted 48 h post-transfection Computerized versions of electropherograms are shown as well as a histogram with standard deviations derived from biologicaltriplicates Primers used for SMN2 amplification were 54C618 (sense) 50-CTCCCATATGTCCAGATTCTCTT-30 and 541C1120 (antisense)50-CTACAACACCCTTCTCACAG-30 The position structure and size of the amplification products corresponding to the splice variants areshown The identity of the doublet bands () migrating above the two SMN2 splice products is unknown The graph displays the inclusion frequency(in percentage) of SMN2 exon 7 based on biological triplicates (C) Expression of the SMN2 protein was analyzed in one set by western blot 72 hafter transfection with using anti-human SMN antibodies (BD Transduction Labs)

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stimulation correlating with increased U1 snRNP bindingat the targeted 50ss The potential of TDP-43-binding tailswas then validated by showing that TDP-43 binding sitesin the tail of a bifunctional oligonucleotide complemen-tary to a region downstream of SMN2 exon 7 shiftedsplicing to favor exon inclusion

In conclusion our studies have increased the variety oftools that can be used alone or in combination with RNAiapproaches to investigate the function of splice variantsor to repress or improve the use of specific splice sitesassociated with a disease TOSS or TOES carrying20OMe tails provide a practical approach that specificallyredirect splicing with no apparent toxic impact therebykeeping cells in a state where they can benefit from anewly acquired function or display information on a newphenotype imposed by the altered production of splicevariants

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online

ACKNOWLEDGEMENTS

We thank Johanne Toutant and Sonia Couture for helpwith tissue culture and extract preparation We thankDouglas Black for Dup 51 Jocelyn Cote for the SMApatient cell line Emanuele Buratti and Francisco Barallefor the TDP-43 antibody and Andres Aguilera for pTER+and pCDNA 6TR plasmids used to produce the inducibleshRNA against TDP-43

FUNDING

This research project was supported by CIHR grants[MOP-272013 to SAE and MOP-93791 to BC] theChaire de recherche de lrsquoUniversite de Sherbrooke onRNA Structure and Genomics (to J-PP) the CanadaResearch Chair in RNA Biology and Cancer Genomics(to SAE) the Canada Research Chair in FunctionalGenomics (to BC) members of the Centre drsquoexcellencede lrsquoUniversite de Sherbrooke en biologie de lrsquoARN (toBC SAE and J-PP) Funding for open access chargeCanadian Institute for Health Research

Conflict of interest statement None declared

REFERENCES

1 PanQ ShaiO LeeLJ FreyBJ and BlencoweBJ (2008)Deep surveying of alternative splicing complexity in the humantranscriptome by high-throughput sequencing Nat Genet 401413ndash1415

2 WangET SandbergR LuoS KhrebtukovaI ZhangLMayrC KingsmoreSF SchrothGP and BurgeCB (2008)Alternative isoform regulation in human tissue transcriptomesNature 456 470ndash476

3 DjebaliS DavisCA MerkelA DobinA LassmannTMortazaviA TanzerA LagardeJ LinW SchlesingerF et al(2012) Landscape of transcription in human cells Nature 489101ndash108

4 NilsenTW and GraveleyBR (2010) Expansion ofthe eukaryotic proteome by alternative splicing Nature 463457ndash463

5 TaziJ BakkourN and StammS (2009) Alternative splicing anddisease Biochim Biophys Acta 1792 14ndash26

6 VenablesJP KlinckR KohC Gervais-BirdJ BramardAInkelL DurandM CoutureS FroehlichU LapointeE et al(2009) Cancer-associated regulation of alternative splicingNat Struct Mol Biol 16 670ndash676

7 Lopez-BigasN AuditB OuzounisC ParraG and GuigoR(2005) Are splicing mutations the most frequent cause ofhereditary disease FEBS Lett 579 1900ndash1903

8 ParkJW PariskyK CelottoAM ReenanRA andGraveleyBR (2004) Identification of alternative splicingregulators by RNA interference in Drosophila Proc Natl AcadSci USA 101 15974ndash15979

9 DominskiZ and KoleR (1993) Restoration of correct splicingin thalassemic pre-mRNA by antisense oligonucleotidesProc Natl Acad Sci USA 90 8673ndash8677

10 ZammarchiF de StanchinaE BournazouE SupakorndejTMartiresK RiedelE CorbenAD BrombergJF andCartegniL (2011) Antitumorigenic potential of STAT3alternative splicing modulation Proc Natl Acad Sci USA 10817779ndash17784

11 Garcia-BlancoMA (2006) Alternative splicing therapeutic targetand tool Prog Mol Subcell Biol 44 47ndash64

12 KoleR KrainerAR and AltmanS (2012) RNA therapeuticsbeyond RNA interference and antisense oligonucleotidesNat Rev Drug Discov 11 125ndash140

13 SinghNK SinghNN AndrophyEJ and SinghRN (2006)Splicing of a critical exon of human Survival Motor Neuron isregulated by a unique silencer element located in the last intronMol Cell Biol 26 1333ndash1346

14 HuaY VickersTA BakerBF BennettCF and KrainerAR(2007) Enhancement of SMN2 exon 7 inclusion by antisenseoligonucleotides targeting the exon PLoS Biol 5 e73

15 GallagherTL ArribereJA GeurtsPA ExnerCRMcDonaldKL DillKK MarrHL AdkarSS GarnettATAmacherSL et al (2011) Rbfox-regulated alternative splicing iscritical for zebrafish cardiac and skeletal muscle functionsDev Biol 359 251ndash261

16 HuaY SahashiK HungG RigoF PassiniMA BennettCFand KrainerAR (2010) Antisense correction of SMN2 splicingin the CNS rescues necrosis in a type III SMA mouse modelGenes Dev 24 1634ndash1644

17 HuaY SahashiK RigoF HungG HorevG BennettCFand KrainerAR (2011) Peripheral SMN restoration is essentialfor long-term rescue of a severe spinal muscular atrophy mousemodel Nature 478 123ndash126

18 HuaY VickersTA OkunolaHL BennettCF andKrainerAR (2008) Antisense masking of an hnRNP A1A2intronic splicing silencer corrects SMN2 splicing in transgenicmice Am J Hum Genet 82 834ndash848

19 SpitaliP and Aartsma-RusA (2012) Splice modulating therapiesfor human disease Cell 148 1085ndash1088

20 CirakS FengL AnthonyK Arechavala-GomezaV TorelliSSewryC MorganJE and MuntoniF (2012) Restoration of thedystrophin-associated glycoprotein complex after exon skippingtherapy in Duchenne muscular dystrophy Mol Ther 20462ndash467

21 LuQL RabinowitzA ChenYC YokotaT YinH AlterJJadoonA Bou-GhariosG and PartridgeT (2005) Systemicdelivery of antisense oligoribonucleotide restores dystrophinexpression in body-wide skeletal muscles Proc Natl Acad SciUSA 102 198ndash203

22 WuB MoultonHM IversenPL JiangJ LiJ LiJSpurneyCF SaliA GuerronAD NagarajuK et al (2008)Effective rescue of dystrophin improves cardiac function indystrophin-deficient mice by a modified morpholino oligomerProc Natl Acad Sci USA 105 14814ndash14819

23 VillemaireJ DionI ElelaSA and ChabotB (2003)Reprogramming alternative pre-messenger RNA splicing throughthe use of protein-binding antisense oligonucleotides J BiolChem 278 50031ndash50039

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24 GendronD CarrieroS GarneauD VillemaireJ KlinckRElelaSA DamhaMJ and ChabotB (2006) Modulation of 50

splice site selection using tailed oligonucleotides carrying splicingsignals BMC Biotechnol 6 5

25 DicksonA OsmanE and LorsonCL (2008) A negativelyacting bifunctional RNA increases survival motor neuron bothin vitro and in vivo Hum Gene Ther 19 1307ndash1315

26 Martinez-ContrerasR CloutierP ShkretaL FisetteJFRevilT and ChabotB (2007) hnRNP proteins and splicingcontrol Adv Exp Med Biol 623 123ndash147

27 Garcia-BlancoMA BaraniakAP and LasdaEL (2004)Alternative splicing in disease and therapy Nat Biotechnol 22535ndash546

28 SkordisLA DunckleyMG YueB EperonIC andMuntoniF (2003) Bifunctional antisense oligonucleotides providea trans-acting splicing enhancer that stimulates SMN2 geneexpression in patient fibroblasts Proc Natl Acad Sci USA100 4114ndash4119

29 CartegniL and KrainerAR (2003) Correction of disease-associated exon skipping by synthetic exon-specific activatorsNat Struct Biol 10 120ndash125

30 MarquisJ MeyerK AngehrnL KampferSSRothen-RutishauserB and SchumperliD (2007) Spinal muscularatrophy SMN2 pre-mRNA splicing corrected by a U7 snRNAderivative carrying a splicing enhancer sequence Mol Ther 151479ndash1486

31 VenablesJP BrosseauJP GadeaG KlinckR PrinosPBeaulieuJF LapointeE DurandM ThibaultP TremblayKet al (2013) RBFOX2 is an important regulator of mesenchymaltissue-specific splicing in both normal and cancer tissuesMol Cell Biol 33 396ndash405

32 PrinosP GarneauD LucierJF GendronD CoutureSBoivinM BrosseauJP LapointeE ThibaultP DurandMet al (2011) Alternative splicing of SYK regulates mitosis andcell survival Nat Struct Mol Biol 18 673ndash679

33 BrosseauJP LucierJF LapointeE DurandM GendronDGervais-BirdJ TremblayK PerreaultJP and ElelaSA (2010)High-throughput quantification of splicing isoforms RNA 16442ndash449

34 KlinckR BramardA InkelL Dufresne-MartinGGervais-BirdJ MaddenR PaquetER KohC VenablesJPPrinosP et al (2008) Multiple alternative splicing markers forovarian cancer Cancer Res 68 657ndash663

35 ChabotB BlanchetteM LapierreI and La BrancheH (1997)An intron element modulating 50 splice site selection in thehnRNP A1 pre-mRNA interacts with hnRNP A1 Mol CellBiol 17 1776ndash1786

36 DignamJD LebovitzRM and RoederRG (1983) Accuratetranscription initiation by RNA polymerase II in a soluble extractfrom isolated mammalian nuclei Nucleic Acids Res 11 1475ndash1489

37 BrosseauJ-P (2012) PhD Thesis Universite de SherbrookeDetection annotation fonctionnelle et regulation des isoformes delrsquoepissage alternatif associees au cancer de lrsquoovaire

38 MarkhamNR and ZukerM (2008) UNAFold software fornucleic acid folding and hybridization Methods Mol Biol 4533ndash31

39 HornungV Guenthner-BillerM BourquinC AblasserASchleeM UematsuS NoronhaA ManoharanM AkiraSde FougerollesA et al (2005) Sequence-specific potent inductionof IFN-alpha by short interfering RNA in plasmacytoid dendriticcells through TLR7 Nat Med 11 263ndash270

40 JudgeAD SoodV ShawJR FangD McClintockK andMacLachlanI (2005) Sequence-dependent stimulation of themammalian innate immune response by synthetic siRNANat Biotechnol 23 457ndash462

41 FedorovY AndersonEM BirminghamA ReynoldsAKarpilowJ RobinsonK LeakeD MarshallWS andKhvorovaA (2006) Off-target effects by siRNA can induce toxicphenotype RNA 12 1188ndash1196

42 PeiY and TuschlT (2006) On the art of identifying effectiveand specific siRNAs Nat Methods 3 670ndash676

43 AltschulSF GishW MillerW MyersEW and LipmanDJ(1990) Basic local alignment search tool J Mol Biol 215403ndash410

44 Thierry-MiegD and Thierry-MiegJ (2006) AceView acomprehensive cDNA-supported gene and transcripts annotationGenome Biol 7(Suppl 1) S1211ndash14

45 StadlerMB ShomronN YeoGW SchneiderA XiaoX andBurgeCB (2006) Inference of splicing regulatory activities bysequence neighborhood analysis PLoS Genet 2 e191

46 BurattiE and BaralleFE (2001) Characterization and functionalimplications of the RNA binding properties of nuclear factorTDP-43 a novel splicing regulator of CFTR exon 9 J BiolChem 276 36337ndash36343

47 SimardMJ and ChabotB (2000) Control of hnRNP A1alternative splicing an intron element represses use of thecommon 30 splice site Mol Cell Biol 20 7353ndash7362

48 AlloM BuggianoV FededaJP PetrilloE SchorI de laMataM AgirreE PlassM EyrasE ElelaSA et al (2009)Control of alternative splicing through siRNA-mediatedtranscriptional gene silencing Nat Struct Mol Biol 16717ndash724

49 Martinez-ContrerasR FisetteJF NasimFU MaddenRCordeauM and ChabotB (2006) Intronic binding sites forhnRNP AB and hnRNP FH proteins stimulate pre-mRNAsplicing PLoS Biol 4 e21

50 NasimFU HutchisonS CordeauM and ChabotB (2002)High-affinity hnRNP A1 binding sites and duplex-forminginverted repeats have similar effects on 50 splice site selection insupport of a common looping out and repression mechanismRNA 8 1078ndash1089

51 BlanchetteM and ChabotB (1999) Modulation of exonskipping by high-affinity hnRNP A1-binding sites and by intronelements that repress splice site utilization EMBO J 181939ndash1952

52 PassoniM De ContiL BaralleM and BurattiE (2012)UG repeatsTDP-43 interactions near 50 splice sites exertunpredictable effects on splicing modulation J Mol Biol 41546ndash60

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