genes & development - a cgenesdev.cshlp.org/content/suppl/2018/03/13/gad.309146... · 2018. 3....

Cas9 recognition site

CA

B

Knuckles_Lence309146_FigS1

1.00E+001.00E+011.00E+021.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+091.00E+10

Me1

l3

Me1

l14

Virm

a

Wtap

Rbm15

Zc3h

13

Hakai

log10iBAQ

Mettl3 IP1 Mean iBAQ A

Enrichment log2 (bait/control)

-log1

0 p-

valu

es

Mettl3 to Control 150mM B


A

- RNase

α-Flag

+ RNase

FlagMyc – Flacc

FlagMyc - Ctr + - + - + -- + - + - +

2% Input IP (Myc)

HA-Fl(2)d + + + + + +

α-HAFl(2)d

Flacc

- RNase

α-Flag

+ RNase

FlagMyc – Flacc

FlagMyc - Ctr + - + - + -- + - + - +

2% Input IP (Myc)

HA-Vir + + + + + +

α-HAVir

Flacc

B

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e tr

ansc

ript l

evel

sFlacc

Flacc KD

Ctr KD

Ctr

– G

FP

Myc

RIP

(M

yc)

Ctr KD Flacc KD

2% In

put

Fl(2

)d –

GF

PM

yc

Nito

– G

FP

Myc

Yth

dc1

– G

FP

Myc

Ctr

– G

FP

Myc

Fl(2

)d –

GF

PM

yc

Nito

– G

FP

Myc

Yth

dc1

– G

FP

Myc

C D


Tunicata; Ascidiacea; …; Ciona intestinalis

Ephydroidea; Drosophilidae; …; Drosophila melanogasterTephritoidea; Tephritidae; …; Ceratitis capitata

Nematocera; Culicomorpha; …; Anopheles gambiae

Echinodermata; Eleutherozoa; …; Strongylocentrotus purpuratus

Chaldicoidea group; …; Pteromalinae; Nasonia vitripennis

Vespoidea; Formicidae; …; Camponotus floridanus

Megachilidae; Megachilinae; …; Megachile rotundataApidae; Apinae; …; Apis mellifera

Papilionoidea; Papilionidae; …; Papilio machaonBombycoidea; Bombycidae; …; Bombyx mori

Hemichordata; Enteropneusta; …; Saccoglossus kowalevskii

Lophotrochozoa; Annelida; …; Capitella teleta

Cephalochordata; Branchiostomidae; Branchiostoma floridae

Cnidaria; Anthozoa; …; Nematostella vectensis

Craniata; Vertebrata

Nematoda

No proteinShort zinc fingerLong zinc fingerMissing zinc finger

Eumetazoa

Bilateria Deuterostomia

Protostomia

Ecdysozoa

Chordata

Panarthropoda; … ; EndopterygotaDiptera

Hymenoptera; Apocrita

Amphiesmenoptera; […]; Obtectomera

Brachycera; …; Acalyptratae

Aculeata

Apoidea

Placozoa

MetazoaOpisthokonta

Fungi

Choanoflagellida

A

C

B C-x8-C-x5-C-x3-H

LOC100186678 (C.i.)LOC100891806 (S.p.)NEMVEDRAFT_v1g24058 (N.v.)CAPTEDRAFT_222693 (C.t.)BRAFLDRAFT_120702 (B.f.)LOC100374861 (S.k.)zc3h13 (D.r.)zc3h13 (C.m.)ZC3H13 (L.c.)zc3h13 (A.c.)ZC3H13 (O.a.)ZC3H13 (G.g.)Zc3h13 (M.m.)ZC3H13 (H.s.)CG7358 (Flacc) (D.m.)Cby1 (C.c.)AgaP_AGAP013495 (A.g.)LOC106713818 (P.m.)LOC101744864 (B.m.)LOC100679030 (N.v.)LOC105254639 (C.f.)LOC100875329 (M.r.)LOC552765 (A.m.)

C-x8-C-x5-C-x5-H* * * *

* * * *

Short zinc finger

Long zinc finger


0.0

0.2

0.4

0.6

0.8

1.0

1.2

Ctr

Met

tl3, M

ettl1

4

Ctr

Met

tl3, M

ettl1

4

Ctr

fl(2)

d

Ctr vir

Ctr

nito Ctr

flaccR

elat

ive

expr

essi

on le

vels

dsRNA

A

0.0

0.2

0.4

0.6

0.8

1.0

Ctr

Met

tl3, M

ettl1

4fla

cc

0.0

0.2

0.4

0.6

0.8

1.0

Ctr

Met

tl3, M

ettl1

4fla

cc

CG8929 (long)CG8929 (short)

dorsal (long)dorsal (short)

0.0

0.2

0.4

0.6

0.8

1.0

Ctr

Met

tl3, M

ettl1

4fla

cc

fl(2)d (long)fl(2)d (short)

Rel

ativ

e is

ofor

m r

atio

(m

RN

A)

dsRNA

C

dMettl3nito

(2)d

virFlacc

dMettl14dMettl3

nito(2)d

virFlacc

dMettl14dMettl3

nito

(2)dvirFlacc

dMettl14

dMettl3

nito(2)d

virFlaccdMettl14

dMettl3

nito

(2)d

virFlacc

dMettl14

dMettl3

nito(2)d

vir

Flacc

dMettl14

1

100

10000

Ctrl

Met

tl3_d

Met

ll14

Fl2d Vir

Nito

Flac

c

Condition

avg.

rpkm

ConditionCtrlMettl3_dMetll14Fl2dVirNitoFlacc

1000

100

10

Ave

rage

rpk

m

Control KD Mettl3, Mettl14 KD

Fl(2)d KD vir KD nito KD Flacc KD

α-Mettl3

α-Mettl14

α-Tubulin

α-Fl(2)d

Ctr

KD

1

Ctr

KD

2

Fla

cc K

D1

Fla

cc K

D2

100

80

80

46

46

B

D E

0.00.20.40.60.81.01.2

Ctr

KD

1

Ctr

KD

2

Fla

cc K

D1

Fla

cc K

D2

Mettl3

0.00.20.40.60.81.01.2

Ctr

KD

1

Ctr

KD

2

Fla

cc K

D1

Fla

cc K

D2

Mettl14

0.0

0.5

1.0

1.5

2.0

Ctr

KD

1

Ctr

KD

2

Fla

cc K

D1

Fla

cc K

D2

fl(2)d

0.00.20.40.60.81.01.2

Ctr

KD

1

Ctr

KD

2

Fla

cc K

D1

Fla

cc K

D2

nito

0.00.20.40.60.81.01.2

Ctr

KD

1

Ctr

KD

2

Fla

cc K

D1

Fla

cc K

D2

vir

0.00.20.40.60.81.01.2

Ctr

KD

1

Ctr

KD

2

Fla

cc K

D1

Fla

cc K

D2

flacc

Rel

ativ

e tr

ansc

ript l

evel

sR

elat

ive

tran

scrip

t lev

els


negative regulation of cellular macromolecule biosynthetic processnegative regulation of macromolecule biosynthetic process

intercellular bridge organizationcell part morphogenesis

plasma membrane bounded cell projection morphogenesiscell projection morphogenesis

neuron projection morphogenesisgermline ring canal formation

cell morphogenesis involved in di erentiationcell morphogenesis involved in neuron di erentiation

0 3 6 9

1e-052e-053e-054e-055e-056e-05

pvalue

10 enriched terms for Fl2d_Vir_Nito_CG7358 common_mis_splice semantic similarity reduced

p-value






0 3 6 9

1e-052e-053e-054e-055e-056e-05

pvalue







0 3 6 9

1e-052e-053e-054e-055e-056e-05

pvalue


0 3 6 9

Enriched terms (Common differentially spliced genes)

Mutually exclusive exon

41

3

1

1

1

0

0

1

00 0

0

0

0

Fl2d Vir

Nito Flacc

overlap of MXE events in commonly spliced genes

122

0

1

0

23

4

0

1

13 4

2

1

1

Fl2d Vir

Nito Flacc

overlap of A5SS events in commonly spliced genesAlternative 5’ ss

316

5

6

4

47

5

0

5

14 6

6

4

1

Fl2d Vir

Nito Flacc

overlap of splice events in commonly spliced genesAll splice events in commonly spliced genes

132

1

3

2

4

0

0

1

00 1

3

3

0

Fl2d Vir

Nito Flacc

overlap of SE events in commonly spliced genes

Exon skipping

21

0

0

1

15

1

0

2

01 1

0

0

0

Fl2d Vir

Nito Flacc

overlap of RI events in commonly spliced genes

Intron retention

Alternative 3’ ss

00

1

1

0

4

0

0

0

00 0

1

0

0

Fl2d Vir

Nito Flacc

overlap of A3SS events in commonly spliced genes

A

0100200300400500600700800

Met

tl3, M

ettl1

4 K

DF

l(2)d

KD

Vir

KD

Nito

KD

Fla

cc K

D

Differentially spliced genes (n)

Nito KD

Mettl3, Mettl14 KD Fl(2)d KD

Vir KD Flacc KD

Control

CG7358 KD! Alternative 3'ss!

Alternative 5'ss!

Mutually exclusive exon!

Intron retention !

Exon skipping!

CB

D E

Commondifferentially

spliced genes(45)

m6A(4018)

3981 37 8

p=1.3e-8


B

C

Rel

ativ

e ex

pres

sion

leve

ls

m6 A

/A r

atio

(m

RN

A)

AFlacc Barentsz DAPI Merge

0

0.02

0.04

0.06

0.08

0.1

0.0

0.5

1.0

1.5

2.0

2.5

embr

yo 0

-1 h

embr

yo 1

-2 h

embr

yo 2

-4 h

embr

yo 4

-6 h

embr

yo 6

-8 h

embr

yo 8

-10

h

embr

yo 1

0-12

h

embr

yo 1

2-14

h

embr

yo 1

4-16

h

embr

yo 1

6-18

h

embr

yo 1

8-20

h

embr

yo 2

0-22

h

L1 la

rvae

L2 la

rvae

youn

g L3

larv

ae

mid

dle

L3 la

rvae

late

L3

larv

ae

whi

te p

repu

pae

Wp

+ 2

4 h

Wp

+ 4

8 h

Wp

+ 7

2 h

adul

t mal

e

adul

t fem

ale

head

s fe

mal

e

ovar

ies

flacc

m6A/A levels

0.00.20.40.60.81.01.21.4

Ctr

KD

Fl(2

)d K

D

fl(2)d

0.00.20.40.60.81.01.21.4

Ctr

KD

Fla

cc K

D

(GD

3521

2)F

lacc

KD

(K

K11

0253

flacc

0.00.20.40.60.81.01.21.4

Ctr

KD

Nito

KD

nito

Rel

ativ

e tr

ansc

ript l

evel

s

* ***(p=3.64E-5)***(p=5.54E-8)

***

D

Stage 2-3(1 - 1:20 h)

Stage 5(2:10 – 2:50 h)

Stage 8(3:10 – 3:40 h)

Stage 13-14(9:20 - 11:20 h)

elav-as elav-s flacc-as flacc-s


A

Ctr KD

α-Myc

Flacc KD

GFPMyc – Mettl3

GFPMyc – Ctr + - + -- + - +

IP (Myc)

HA-Mettl14 + + + +

α-HA

Mettl3

Mettl14

+ - + -- + - +

2% input

+ + + +

Ctr KD Flacc KD

*

C D

B

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Rel

ativ

e tr

ansc

ript l

evel

s

Flacc

Flacc KDCtr KD

F

0.00.20.40.60.81.01.2

Ctr

FM

-Vir

HA

Nito

FM

-Vir

HA

Ctr

FM

-Vir

HA

Nito

FM

-Vir

HA

Rel

ativ

e tr

ansc

ript l

evel

s

Flacc

Flacc KDCtr KD

Ctr KD

α-Myc

Flacc KD

GFPMyc – Nito

GFPMyc – Ctr + - + -- + - +

IP (Myc)

HA-Vir + + + +

α-HA

Nito

Vir

+ - + -- + - +

2% input

+ + + +

Ctr KD Flacc KD

E

α-Myc

Myc-Fl(2)d short

GFPMyc–Ctr

Myc-Fl(2)d long

+ - - +

- + - -

2% input

- - + -

- -

+ -

- +

HA-Vir + + + + + +

α-HA

Ctr KD Flacc KD Ctr KD Flacc KD

Fl(2)d short

Fl(2)d long

Vir

+ - - +

- + - -

IP (Myc)

- - + -

- -

+ -

- +

+ + + + + +

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Ctr

FM

-Vir

HA

Fl(2

)d s

hort

FM

-Vir

HA

Fl(2

)d lo

ng F

M-V

ir H

AC

tr F

M-V

ir H

AF

l(2)d

sho

rt F

M-V

ir H

AF

l(2)d

long

FM

-Vir

HAR

elat

ive

tran

scrip

t lev

els

Flacc

Flacc KDCtr KD

G

H

α-Myc

Ctr KD Flacc KD

FlagMyc – Nito

FlagMyc – Ctr + - + -- + - +

IP (Myc)

HA – Mettl3 + + + +

α-HAMettl3

+ - + -- + - +

2% input

+ + + +

Ctr KD Flacc KD

Nito

Ctr KD

α-Myc

Flacc KD

FlagMyc – Nito

FlagMyc – Ctr + - + -- + - +

IP (Myc)

HA – Mettl14 + + + +

α-HAMettl14

+ - + -- + - +

2% input

+ + + +

Ctr KD Flacc KD

Nito

0.00.20.40.60.81.01.21.4

Ctr

GF

PM

yc-F

l(2)d

HA

Nito

GF

PM

yc-F

l(2)d

HA

Ctr

GF

PM

yc-F

l(2)d

HA

Nito

GF

PM

yc-F

l(2)d

HA

Rel

ativ

e tr

ansc

ript l

evel

s

Flacc

Flacc KDCtr KD

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1 2 3 4 5 6 7 8

Rel

ativ

e tr

ansc

ript l

evel

s

Flacc

Flacc KDCtr KD

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

3 4 7 8

Rela&velevelsofZC3

H13

I

J K


0.00E+00

2.00E+04

4.00E+04

6.00E+04

8.00E+04

1.00E+05

1.20E+05

empt

y

empt

y

Wta

p-C

-sm

+ R

bm15

-C-lg

Wta

p-C

-sm

+ R

bm15

-N-lg

Wta

p-C

-lg +

Rbm

15-C

-sm

Wta

p-C

-lg +

Rbm

15-N

-sm

Wta

p-N

-sm

+ R

bm15

-C-lg

Wta

p-N

-sm

+ R

bm15

-N-lg

Wta

p-N

-lg +

Rbm

15-C

-sm

Wta

p-N

-lg +

Rbm

15-N

-sm

RL

URbm15-Wtap NanoBiT

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

WT

Zc3

h13

KO

Met

tl3 K

ORel

ativ

e m

RN

A n

orm

aliz

ed t

o G

AP

DH

NanoBiT fusion expression level

Wtap-N-sm

Rbm15-C-lg

B

total spectrum count/ total unique peptide count/ % sequence coverage

Rbm15 IP-LC-MS from nuclear fraction

enrichment log2 (bait/control)

-log1

0 p-

valu

esRbm15 WT to Rbm15 in Zc3h13 KOA C

D E F


genes & development - a cgenesdev.cshlp.org/content/suppl/2018/03/13/gad.309146... · 2018. 3....

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