genes & development - a cgenesdev.cshlp.org/content/suppl/2018/03/13/gad.309146... · 2018. 3....
TRANSCRIPT
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
Knuckles_Lence309146_FigS2
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
Knuckles_Lence309146_FigS3
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
Knuckles_Lence309146_FigS4
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
Knuckles_Lence309146_FigS5
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
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
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
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
Knuckles_Lence309146_FigS6
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
Knuckles_Lence309146_FigS7
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
Knuckles_Lence309146_FigS8
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
Knuckles_Lence309146_FigS9