TncA

Yano_Supplemental_FigS8

skipped

spliced

Cont. shQk

1 2 3 1 2 3

skipped

spliced

Cont. Qk cKOHet WT1 WT2 KO1 KO2 KO3

BPkm1

Pkm2

Cont.shQk

Pkm2

R²=0.91051

0

1

2

3

4

5

0 0.2 0.4 0.6 0.8 1 1.2Relativ

eexpressio

n(Pkm

1)

Relativeexpression(Qk)

siQk1siQk2

siQk3

Cont.

pstI cut

C

Yano_Supplemental_FigS8

F

Scale:chr13

STS Markers

Other RefSeqRefSeq GenesSequences

Spliced ESTs

RatHumanOrangutanDogHorseOpossumChickenSticklebackRepeatMaskerSNPs (128)

5 kbmm958,495,00058,500,000

Q5

Q(-) description= Q Cluster Density

STS Markers on Genetic and Radiation Hybrid MapsUCSC Genes (RefSeq, GenBank, tRNAs & Comparative Genomics)

Ensembl Gene Predictions - archive 65 - dec2011

Human Proteins Mapped by Chained tBLASTn

Non-Mouse RefSeq GenesRefSeq Genes

Publications: Sequences in Scientific ArticlesMouse mRNAs from GenBank

Mouse ESTs That Have Been SplicedPlacental Mammal Basewise Conservation by PhyloP

Multiz Alignments of 30 Vertebrates

Repeating Elements by RepeatMaskerSimple Nucleotide Polymorphisms (dbSNP build 128)

HnrnpkHnrnpkHnrnpkHnrnpkHnrnpkHnrnpkHnrnpk

Mir7-1Hnrpk

Rmi1

ENSMUST00000116403ENSMUST00000116404ENSMUST00000172426ENSMUST00000169846ENSMUST00000109849ENSMUST00000043269ENSMUST00000083500

HNRPKHNRPK

AK160887BC089328AK135519AK166656AK168918AK166927AK151689AK152927AK169064AK167600AK128891AK150848AK088462BC006694AK051313AK011428AK078777AK160615AK206192AK196607AK192668AK186860AK211652AK215933AK203272AK207611AK204979EU604547

DM176671

AK208657AK205281

AK179362AK197677

AK201304AK191331AK201722AK204853AK220023

AK194621AK028023

AK079457

AK139703

n= Q Cluster Density_ 93

1

Mammal Cons

_ 2.1

-3.3

- 0

Scale:chr13

STS Markers

Other RefSeqRefSeq GenesSequences

Spliced ESTs

RatHumanOrangutanDogHorseOpossumChickenSticklebackRepeatMaskerSNPs (128)

500 basesmm958,494,00058,494,500

Q5

Q(-) description= Q Cluster Density

STS Markers on Genetic and Radiation Hybrid MapsUCSC Genes (RefSeq, GenBank, tRNAs & Comparative Genomics)

Ensembl Gene Predictions - archive 65 - dec2011

Human Proteins Mapped by Chained tBLASTn

Non-Mouse RefSeq GenesRefSeq Genes

Publications: Sequences in Scientific ArticlesMouse mRNAs from GenBank

Mouse ESTs That Have Been SplicedPlacental Mammal Basewise Conservation by PhyloP

Multiz Alignments of 30 Vertebrates

Repeating Elements by RepeatMaskerSimple Nucleotide Polymorphisms (dbSNP build 128)

HnrnpkHnrnpkHnrnpkHnrnpkHnrnpkHnrnpkHnrnpk

Mir7-1ENSMUST00000116403ENSMUST00000116404ENSMUST00000172426ENSMUST00000169846ENSMUST00000109849ENSMUST00000043269

ENSMUST00000083500HNRPKHNRPK

AK160887BC089328AK135519AK166656AK168918AK166927AK151689AK152927AK169064AK167600AK128891AK150848AK088462BC006694AK051313AK011428AK078777AK160615AK206192AK196607AK192668AK186860AK211652AK215933AK203272AK207611AK204979

EU604547DM176671

n= Q Cluster Density_ 93

1

Mammal Cons

_ 2.1

-3.3

- 0

Scale:chr13--->

STS Markers

Other RefSeqRefSeq GenesSequences

Spliced ESTs

GapsMouseRatHumanOrangutanDogHorseOpossumChickenSticklebackRepeatMaskerSNPs (128)

20 basesmm958,494,34058,494,35058,494,36058,494,37058,494,38058,494,39058,494,400

TCTTCAAACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTTACAGAAGTAAAATCATTCTTTTGTCTTCTTTTAATTQ5

Q(-) description= Q Cluster Density

STS Markers on Genetic and Radiation Hybrid MapsUCSC Genes (RefSeq, GenBank, tRNAs & Comparative Genomics)

Ensembl Gene Predictions - archive 65 - dec2011

Human Proteins Mapped by Chained tBLASTnNon-Mouse RefSeq Genes

RefSeq GenesPublications: Sequences in Scientific Articles

Mouse mRNAs from GenBank

Mouse ESTs That Have Been SplicedPlacental Mammal Basewise Conservation by PhyloP

Multiz Alignments of 30 Vertebrates

Repeating Elements by RepeatMaskerSimple Nucleotide Polymorphisms (dbSNP build 128)

HnrnpkHnrnpkHnrnpkHnrnpkHnrnpkHnrnpkHnrnpkENSMUST00000116403ENSMUST00000116404ENSMUST00000172426ENSMUST00000169846ENSMUST00000109849ENSMUST00000043269

AK160887BC089328AK135519AK166656AK168918AK166927AK151689AK152927AK169064AK167600AK128891AK150848AK088462BC006694AK051313AK011428AK078777AK160615AK211652AK215933AK203272AK207611AK204979EU604547

2171TCTTCAAACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTTACAGAAGTAAAATCATTCTTTTGTCTTCTTTTAATTTCTTCAAACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTTACAGAAGTAAAATCATTCTTTTGTCTTCTTTTAATTTCTTCAAACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTTACAGAAGTAAAATCATTCTTTCGTCTTCTTTTAATTTCTTCAAACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTTACAGAAGCAAAATCATTCTTTCGTCTTCTTTTAATTTCTTCAAACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTTACAGAAGCAAAATCATTCTTTCGTCTTCTTTTAATTTCTTCAAACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTTACAGAAGCAAAATCATTCTTTCGTCCTCTTTTAATTTCTCCTGACGAACCAATTCCGAAGAAGGTCGTTCTAATCATAACTCACAGAAGTAAAATCATTCTTTCGTCTTCTTTTA-CTTCTCCTGACGAACCAATTTCACAGAAGGTCGT-TTAATCAAAACTCACAGAAGTAAAATCAATCTTTTGCCTCTTCTTAGTT==================================================================================

n= Q Cluster Density_ 93

1

Mammal Cons

_ 2.1

-3.3

- 0

0.0

0.5

1.0

1.5

2.0

Cont shQkRelativ

elevelofm

iR-7a-1 P=0.016

D

0

1

2

3

4

5

Pea15a Itgb8 Vim Vcam1 Tnc Brsk2 ptpro Nsg2 Chgb

Control QkcKO

apicalRG

Relativeexpressio

n

E

0

2

4

6

8

10

Dpysl5 Ckmt1 Tubb3 Snap25 Thy1 Slc25a18 Mlc1 Cd44 Acsbg1 ilrap1 Pdgfra

Control QkcKO

Neuron Glia

Relativeexpressio

n

basalRG

miR-7-1

Fig.S8Qki5-CLIPcombinedwithtranscriptomeanalysisandbiochemicalvalidationrevealQki5-dependentRNAmetabolism

(A)GenomebrowserviewofQki5CLIPclusterdensityalongtheTnc gene,includingthealternativeexonandtheflankingregion(top-leftpanel).Magnifiedviewofa89-ntregionofTncshowingtheQki5CLIPcluster.HighlightsindicateQk5bindingmotifs(bottom-leftpanel).RT-PCRanalysisofTnc pre-mRNAsplicinginsh-cont andsh-Qk neuralstemcells,whichwasperformedtomonitorthesplicingofanalternativeexon.Primerswereusedthatcorrespondedtoexons14(forward)and15(reverse),andtheresultsindicatetheexpressionofaberrantlyprocessedTncmRNAbothinsh-Qk andQk cKO neuralstemcells(rightpanel).(B)RT-PCRfollowedbyPstI digestiontomonitorpkm1/2 pre-mRNAsplicingofanalternativeexoninsh-cont andsh-Qk neuralstemcells.(C) Scatterplotofthe mRNAexpressionlevelsofpkm1 (Y-axis)andQk (X-axis)inculturedneuralstemcellstransfectedwith3controls(withoutsiRNA,negativecontrolsiRNA1andnegativecontrolsiRNA2,asindicatedbybluedots)and3siRNAsforQk (siQk1,siQk2andsiQk3,asindicatedbygreendots).Thereisastrongnegativecorrelationbetweenpkm1 andQkexpressiondependingontheknock-downefficiencyofQk (R2=0.91).(D) BargraphofQki-dependenttranscriptlevelchangesinaRG-related(pea15a,Itgb8,vim,vcam1andTnc)andbRG-relatedgenes(Brsk2,ptpro,Nsg2andchgb)validatedbyqRT-PCRusingmRNAfromculturedneuralstemcellsofcontrolorQk cKO mice.TranscriptlevelsofaRG-relatedtranscriptsaredecreased,whiletranscriptlevelsofbRG-relatedtranscriptsareincreasedinQk deficient neuralstemcells.ErrorbarrepresentsSDusingthreeindependentbiologicalreplicates. ThedataispresentedasnormalizedexpressionrelativetoYwhaz.(E)BargraphofQki-dependenttranscriptlevelchangesinNeuron-relatedandGlia-relatedgenesvalidatedbyqRT-PCRusingmRNAfromculturedneuralstemcellsofcontrolorQk cKOmice.TranscriptlevelsofGlia-relatedtranscriptsaredecreased,whiletranscriptlevelsofNeuron-relatedtranscriptsareincreasedinQk cKO neuralstemcells.ErrorbarrepresentsSDusingthreeindependentbiologicalreplicates. ThedataispresentedasnormalizedexpressionrelativetoYwhaz.(F)GenomebrowserviewofQki5CLIPclusterdensityalongthehnrnpk gene,includingtheintronic miRNAmiR-7-1 (toppanel).Magnifiedviewofhnrnpk gene showingtheQki5-CLIPclustercenteredupstreamofmiR-7-1 (leftpanel).Magnifiedviewofa72-ntregionofhnrnpkshowingtheQki5-CLIPclustercenteredupstreamofmiR-7-1 (rightpanel).qRT-PCRanalysisofmiR-7-1 insh-cont andSh-Qk wasperformedtomonitortheexpressionlevelofmaturemiRNArelativetoU6 RNA.Theresultsindicatethattheexpressionofmir-7-1isup-regulatedinsh-Qkneuralstemcells(insertleftpanel).