A2CA2GA2TC2AC2GC2T A2CA2GA2TC2AC2GC2T A2CA2GA2TC2AC2GC2T A2CA2GA2TC2AC2GC2T A2CA2GA2TC2AC2GC2T A2CA2GA2TC2AC2GC2T

0.0

0.2

0.4

0.6

Substitution

% A

pre

cedi

ng s

ubst

itutio

n

MutationA2CA2GA2TC2AC2GC2T

Freq. of A preceding the substitution (m/k filter applied)T1 (0 - 45 min) T2 (45 - 90 min) T3 (1.5 - 6 hr) T4 (6 - 12 hr) T5 (12 - 18 hr) T6 (18 - 24 hr)

Supplementary Figure 1. Selective enrichment of A upstream of C-to-T transitions in miCLIP data. The frequency of adenosine preceding C-to-T transitions annotated through the CIMS pipeline is sub-stantially higher than other types of nucleotide substitutions. Note that for the T6 library, the “A” enrichment is not as high as in other libraries, indicating CIMS confidence is less certain.

0

200

400

600

−10 0 10Distance from C −> T

Uni

que

geno

mic

coo

rdin

ates

0

250

500

750

−10 0 10Distance from C −> T

Uni

que

geno

mic

coo

rdin

ates

0

2000

4000

−10 0 10Distance from C −> T

Uni

que

geno

mic

coo

rdin

ates

0

1000

2000

3000

−10 0 10Distance from C −> T

Uni

que

geno

mic

coo

rdin

ates

0

500

1000

1500

2000

−10 0 10Distance from C −> T

Uni

que

geno

mic

coo

rdin

ates

0

500

1000

1500

2000

−10 0 10Distance from C −> T

Uni

que

geno

mic

coo

rdin

ates

AAAAACACTGACGGAothers

AAC

GACAAA AAA

ACT

GGA

AAC

GAC

AAA AAA

ACT

GGA

AAC

GAC

AAA AAA

ACT

GGA

AAC

GACAAA

AAA

ACT

GGA

AAC

GACAAA AAA

ACT

GGA

AAC

GACAAA

AAA

ACT

GGA

Supplementary Figure 2. Positional enrichment of submotifs in the vicinity of CIMS calls. Shown are motif enrichment plots centered on CIMS calls in each library. The peak is shown at the first nucleotide of the motif. GAC, AAC, and ACT, which form submotifs on the canonical DRACH motif of m6A sites in other species. Note that the fly m6A sites show prefer-ence for being in local A-rich regions (AAA submotifs, which are not contingent on location at a CIMS). However, GGA submotif, which is also not contingent on location at a CIMS, is also enriched precisely at the three nucleotides upstream of CIMS.

Timepoint 1 (0 - 45 min)

Timepoint 2 (45 - 90 min)

Timepoint 3 (1.5 - 6 hr)

Timepoint 4 (6 - 12 hr)

Timepoint 5 (12 - 18 hr)

Timepoint 6 (18 - 24 hr)

5’ UTR

CDS

3’ UTR

intron

other

17.9% 24.5% 22.5%

29.9% 30.3% 31.9%

45.2%41.2% 41.2%

35.8% 45.2% 29.8%

20.9%22.4% 20%

19.4%18%

14.3%

0.7% 0.2% 1.3%

1.2%1.8%

4.5%

13.7% 13.1% 15%

13.5% 16.2% 19.3%

Features

Timepoint 1 (0 - 45 min) Timepoint 2 (45 - 90 min) Timepoint 3 (1.5 - 6 hr)

Timepoint 4 (6 - 12 hr) Timepoint 5 (12 - 18 hr) Timepoint 6 (18 - 24 hr)

Supplementary Figure 3. Genomic locations of CIMS calls across the miCLIP datasets. The overall distribution of CIMS calls relative to genomic annotations is similar across embryogenesis, except that the fraction of intronic hits increases with the onset of zygotic transcription (in timepoint 3).

Supplementary Figure 5. Drosophila YTH domains bear conserved features for m6A binding. YTH domains from CG12076/YT521-B (A) and CG6422 (B) have a significant homology with the domains from other YTH proteins that are known to bind m6A. Shown here are amino acid alignments of the two YTH domains with the YTH domains from other organisms. The secondary structure elements from rat YTHDC1 (PDB accession number: 2mtv) and human YTHDF1 (PDB accession number: 4rcj) are also shown at the top of each alignment in panels A and B, respectively. Amino acids interacting with m6A and the RNA backbone are indicated below the alignments with colored circles and squares. Resi-dues forming the hydrophobic pocket are indicated by blue circles, and the residues contacting the RNA are shown in orange or blue squares.

YTH_YTHDC1_R. norvegicus [2mtv]

YTHDC1_H. sapiens [Q96MU7]YTHDC1_R. norvegicus [Q9QY02]

YTHDC1_D. rerio [Q5U3V6]YTHDC1_X. laevis [Q5XGV7]

CG12076_D. melanogaster [Q9VZQ1]

355358334299254

YTH_YTHDC1_R. norvegicus [2mtv]

YTHDC1_H. sapiens [Q96MU7]YTHDC1_R. norvegicus [Q9QY02]

YTHDC1_D. rerio [Q5U3V6]YTHDC1_X. laevis [Q5XGV7]

CG12076_D. melanogaster [Q9VZQ1]

401404380345300

YTH_YTHDC1_R. norvegicus [2mtv]

YTHDC1_H. sapiens [Q96MU7]YTHDC1_R. norvegicus [Q9QY02]

YTHDC1_D. rerio [Q5U3V6]YTHDC1_X. laevis [Q5XGV7]

CG12076_D. melanogaster [Q9VZQ1]

447450426391346

YTH_YTHDF1_H. sapiens [4rcj]

YTHDF1_H. sapiens [Q9BYJ9]YTHDF1_R. norvegicus [Q4V8J6]

YTHDF1_D. rerio [Q802Z0]YTHDF1_X. laevis [Q6PA59]

CG6422_D. melanogaster [Q9VBZ5]

389389395429382

YTH_YTHDF1_H. sapiens [4rcj]

YTHDF1_H. sapiens [Q9BYJ9]YTHDF1_R. norvegicus [Q4V8J6]

YTHDF1_D. rerio [Q802Z0]YTHDF1_X. laevis [Q6PA59]

CG6422_D. melanogaster [Q9VBZ5]

435435441475428

YTH_YTHDF1_H. sapiens [4rcj]

YTHDF1_H. sapiens [Q9BYJ9]YTHDF1_R. norvegicus [Q4V8J6]

YTHDF1_D. rerio [Q802Z0]YTHDF1_X. laevis [Q6PA59]

CG6422_D. melanogaster [Q9VBZ5]

481481487521474

Amino acids forming hydrophobic pocketAmino acids involved in RNA binding

Source of annotations: Theler et al., NAR 2014 (doi: 10.1093/nar/gku1116)

Amino acids forming hydrophobic pocketAmino acids involved in binding non-m6A part of RNA

Source of annotations: Luo and Tong. PNAS 2014 (doi: 10.1073/pnas.1412742111)Amino acids interacting with m6A

A

B

Scale:chr3L+3,370,180-3,371,256-3,374,309

Other RefSeqSpliced ESTs

Cons ElementsD_simulansD_sechelliaD_yakubaD_erectaD_ananassaeD_pseudoobscuraD_persimilisD_willistoniD_virilisD_mojavensisD_grimshawiRepeatMasker

1 kbdm63,370,5003,371,0003,371,5003,372,0003,372,5003,373,0003,373,5003,374,0003,374,500

Perfect Matches to Short Sequence (TACCTC)

Your Sequence from Blat SearchRefSeq Genes (by FlyBase)

Non-D. melanogaster RefSeq GenesD. melanogaster ESTs That Have Been Spliced

D. melanogaster mRNAs from GenBank

Multiz Alignment & Conservation (27 Species)Multiz Alignments of 27 insects

Repeating Elements by RepeatMasker

YourSeqDrsYT521-B

YT521-BCG12010

X75595AY119009FJ632093KX531656KX800279KX803924

BT001450AF145594BT150336

JV215176JV226809

JV212321BT150096

YT521-B[NP1], [NP2], [NP3]

Scale:chr3R

Other RefSeqSpliced ESTs

Cons ElementsD_simulansD_sechelliaD_yakubaD_erectaD_ananassaeD_pseudoobscuraD_persimilisD_willistoniD_virilisD_mojavensisD_grimshawiRepeatMasker

2 kbdm625,033,50025,034,00025,034,50025,035,00025,035,50025,036,00025,036,50025,037,00025,037,50025,038,00025,038,50025,039,00025,039,500

Perfect Matches to Short Sequence (TACCTC)Your Sequence from Blat Search

RefSeq Genes (by FlyBase)

Non-D. melanogaster RefSeq GenesD. melanogaster ESTs That Have Been Spliced

D. melanogaster mRNAs from GenBank

Multiz Alignment & Conservation (27 Species)Multiz Alignments of 27 insects

Repeating Elements by RepeatMasker

YourSeqSmg6CG6422

CG6422CG6422CG6422

CG31115

bai

BT023791JV211305JV228042KX800466AJ556816

BT001679BT133047AY069675

JV214827FJ636306FJ631484KX797630

JV230791JV226565

BT012341BT099648

KX796746

JV221822AY075503

JV214284FJ632761

FJ637112

CG6422

Scalechr3R:

Gap

Conservation

d_simulansd_sechellia

d_yakubad_erecta

d_ananassaed_pseudoobscura

d_persimilisd_willistoni

d_virilisd_mojavensisd_grimshawi

a_gambiaea_mellifera

t_castaneum

1 kb dm319,857,000 19,857,500 19,858,000 19,858,500 19,859,000 19,859,500 19,860,000 19,860,500

RefSeq Genes

Your Sequence from Blat SearchPerfect Matches to Short Sequence (TACCTC)

Gap LocationsFlyBase Protein-Coding Genes

FlyBase Noncoding Genes12 Flies, Mosquito, Honeybee, Beetle Multiz Alignments & phastCons Scores

KrT95DKrT95DKrT95DKrT95D

Ime4 MiroMiroMiroMiro

YourSeq

KrT95DKrT95DKrT95D

CG5933 MiroMiro

mettl3[SK2]

mettl3

WT GCCAACCAGGGTGCAATTGTGATCCGGAACGTGACCATCGGCACCGAGGCCGGCTGCGAAATCATCTCTGTGCAGCCCAAGGAGCTGAAGGAGATCCTGGAAGACACCAACGATACGTGTCAGCAAAAGGAGGAGGAGGCCAAGAGAAAGTGTGGGTCTCACAGAAATTACTACTCTCATAAGTCTAATAA

A N Q G A I V I R N V T I G T E A G C E I I S V Q P K E L K E I L E D T N D T C Q Q K E E E A K R K C G S H R N Y Y S H K S N

SK2 GCCAACCAGGGTGCAATTGTGATCCGGAACGTGACCAccgaGCACCGAGGCCGGCTGCGAAATCATCTCTGTGCAGCCCAAGGAGCTGAAGGAGATCCTGGAAGACACCAACGATACGTGTCAGCAAAAGGAGGAGGAGGCCAAGAGAAAGTGTGGGTCTCACAGAAATTACTACTCTCATAAGTCTAATAA +1

A N Q G A I V I R N V T T E H R G R L R N H L C A A Q G A E G D P G R H Q R Y V S A K G G G G Q E K V W V S Q K L L L S * V * * +48aa

mettl14

wt CAGCACTTTGTGGACACCGGTCAGCGTCCCCAGAACTTCATCCGTGATGTCGGCCTAGCTGACCGCTTCGAGGAGTATCCCAAGTTGAGGGAACTGAT

Q H F V D T G Q R P Q N F I R D V G L A D R F E E Y P K L R E L

SK1 CAGCACTTTGTGGACACCGTCCCCAGAACTTCATCCGTGATGTCGGCCTAGCTGACCGCTTCGAGGAGTATCCCAAGTTGAGGGAACTGAT -7

Q H F V D T V P R T S S V M S A *

SK2 CAGCACTTTGTGGACAgttctgTCCCCAGAACTTCATCCGTGATGTCGGCCTAGCTGACCGCTTCGAGGAGTATCCCAAGTTGAGGGAACTGAT -4

Q H F V D S S V P R T S S V M S A *

Scalechr2L:

Gap

Conservation

d_simulansd_sechellia

d_yakubad_erecta

d_ananassaed_pseudoobscura

d_persimilisd_willistoni

d_virilisd_mojavensisd_grimshawi

a_gambiaea_mellifera

t_castaneum

1 kb dm38,307,900 8,308,000 8,308,100 8,308,200 8,308,300 8,308,400 8,308,500 8,308,600 8,308,700 8,308,800 8,308,900 8,309,000 8,309,100 8,309,200 8,309,300 8,309,400 8,309,500 8,309,600 8,309,700 8,309,800 8,309,900 8,310,000 8,310,100 8,310,200 8,310,300

RefSeq Genes

Your Sequence from Blat SearchPerfect Matches to Short Sequence (TACCTC)

Gap LocationsFlyBase Protein-Coding Genes

FlyBase Noncoding Genes12 Flies, Mosquito, Honeybee, Beetle Multiz Alignments & phastCons Scores

Ostgamma CG7818CG7810

YourSeq+8,308,224

CG7830 CG7818CG7810

mettl14[SK1]mettl14[SK2]

f(l)2d

wt GCGCCAACTGCCCTCGCATTGCGCACTGCTCTGCTCGATCCTGCGGTCAATCTCCTGTTCGAGCGGCTCAAGAAGGAACTTAAGGCCACCAAGGCCAAGCTGGAGGAGACCCAGAACGAGCTGTCCGCATGGAAGTTCACGCCGGACTCTAATACCGGCAAACGCCTAA

A P T A L A L R T A L L D P A V N L L F E R L K K E L K A T K A K L E E T Q N E L S A W K F T P D S N T G K R L

SK2 GCGCCAACTGCCCTCGCACTGCTCTGCTCGATCCTGCGGTCAATCTCCTGTTCGAGCGGCTCAAGAAGGAACTTAAGGCCACCAAGGCCAAGCTGGAGGAGACCCAGAACGAGCTGTCCGCATGGAAGTTCACGCCGGACTCTAATACCGGCAAACGCCTAA -7

A P T A L A L L C S I L R S I S C S S G S R R N L R P P R P S W R R P R T S C P H G S S R R T L I P A N A *

SK4 GCGCCAACTGCACTGCTCTGCTCGATCCTGCGGTCAATCTCCTGTTCGAGCGGCTCAAGAAGGAACTTAAGGCCACCAAGGCCAAGCTGGAGGAGACCCAGAACGAGCTGTCCGCATGGAAGTTCACGCCGGACTCTAATACCGGCAAACGCCTAA -13

A P T A L L C S I L R S I S C S S G S R R N L R P P R P S W R R P R T S C P H G S S R R T L I P A N A *

Scale:chr2R

Other RefSeqSpliced ESTs

Cons ElementsD_simulansD_sechelliaD_yakubaD_erectaD_ananassaeD_pseudoobscuraD_persimilisD_willistoniD_virilisD_mojavensisD_grimshawiRepeatMasker

2 kbdm613,818,50013,819,00013,819,50013,820,00013,820,50013,821,00013,821,50013,822,00013,822,50013,823,00013,823,50013,824,00013,824,50013,825,000

Perfect Matches to Short Sequence (TACCTC)Your Sequence from Blat Search

RefSeq Genes (by FlyBase)

Non-D. melanogaster RefSeq GenesD. melanogaster ESTs That Have Been Spliced

D. melanogaster mRNAs from GenBank

Multiz Alignment & Conservation (27 Species)Multiz Alignments of 27 insects

Repeating Elements by RepeatMasker

YourSeqstjstjstj

fl(2)d

fl(2)dfl(2)dfl(2)d

CG13339

AY069830FJ638683KX803269AY061583JV228963

AY069478AJ243599AJ243607

JV209491JV209490

FJ636472

FJ631637JV228720

JV221327

JV222488

JV229494AY070591BT032653JV210506KX796175KX806407

fl(2)d[SK2]fl(2)d[SK4]

wt CTGCCGATGCGCGAGATGGCGGACTTGGATGCAGTGCACCTGGGCCTCGACGAGAACGAGGCGGACATTGCCGAGGAGCTGCAAGACTTTGAGTTCAACACAAGGAGTGAGGCTTCCGAATCGAATGGTGGAGACTCATCCGACTCGGAGCCGAGCATCAGCTCCGTCAGCACTGCCACATCTTCCCTGGCGGGCAGTAGCAAGCGGAAAACCAAGAAGCCTGCCAAGCAAAGCCCTCAACCCGCTGTCGAGACCAAATCCTCCAAATCTTCCGCCAAGAACAAAGCCAAACGGGAACCCACTCCCGAGGAGCTAAATG

L P Met R E Met A D L D A V H L G L D E N E A D I A E E L Q D F E F N T R S E A S E S N G G D S S D S E P S I S S V S T A T S S L A G S S K R K T K K P A K Q S P Q P A V E T K S S K S S A K N K A K R E P T P E E L N

#R/NP2 CTGCCGATGC-CGAGATGGCGGACTTGGATGCAGTGCACCTGGGCCTCGACGAGAACGAGGCGGACATTGCCGAGGAGCTGCAAGACTTTGAGTTCAACACAAGGAGTGAGGCTTCCGAATCGAATGGTGGAGACTCATCCGACTCGGAGCCGAGCATCAGCTCCGTCAGCACTGCCACATCTTCCCTGGCGGGCAGTAGCAAGCGGAAAACCAAGAAGCCTGCCAAGCAAAGCCCTCAACCCGCTGTCGAGACCAAATCCTCCAAATCTTCCGCCAAGAACAAAGCCAAACGGGAACCCACTCCCGAGGAGCTAAATG -1 out of frame ATG1, ATG2 retained and translatable -1 L P Met P R W R T W Met Q C T W A S T R T R R T L P R S C K T L S S T Q G V R L P N R Met V E T H P T R S R A S A P S A L P H L P W R A V A S G K P R S L P S K A L N P L S R P N P P N L P P R T K P N G N P L P R S * Met

#T/NP3 CTGCCGATGCGCG--------GACTTGGATGCAGTGCACCTGGGCCTCGACGAGAACGAGGCGGACATTGCCGAGGAGCTGCAAGACTTTGAGTTCAACACAAGGAGTGAGGCTTCCGAATCGAATGGTGGAGACTCATCCGACTCGGAGCCGAGCATCAGCTCCGTCAGCACTGCCACATCTTCCCTGGCGGGCAGTAGCAAGCGGAAAACCAAGAAGCCTGCCAAGCAAAGCCCTCAACCCGCTGTCGAGACCAAATCCTCCAAATCTTCCGCCAAGAACAAAGCCAAACGGGAACCCACTCCCGAGGAGCTAAATG -8 out of frame ATG1 and deleted for ATG2 -8 L P Met R G L G C S A P G P R R E R G G H C R G A A R L *

#X/NP1 CTGCCGATGCGCatcGATGGCGGACTTGGATGCAGTGCACCTGGGCCTCGACGAGAACGAGGCGGACATTGCCGAGGAGCTGCAAGACTTTGAGTTCAACACAAGGAGTGAGGCTTCCGAATCGAATGGTGGAGACTCATCCGACTCGGAGCCGAGCATCAGCTCCGTCAGCACTGCCACATCTTCCCTGGCGGGCAGTAGCAAGCGGAAAACCAAGAAGCCTGCCAAGCAAAGCCCTCAACCCGCTGTCGAGACCAAATCCTCCAAATCTTCCGCCAAGAACAAAGCCAAACGGGAACCCACTCCCGAGGAGCTAAATG -2/+3 out of frame ATG1, ATG2 retained and translatable-2/+3 L P Met R I D G G L G C S A P G P R R E R G G H C R G A A R L *

CG6422

wt GTCGAGGAGCGCAACATACCATGGAGTCAGCAAGTCGATGAGGCCTCGTACGAAAATCTATCCTCTCCCACTCACGATGAAGTCTCTGGTGAGTTCTTCGACTATAAGAAATATATGGGTTTCCAAAAGCCATCTAACCCATGTCTATTCCTAACC

V E E R N I P W S Q Q V D E A S Y E N L S S P T H D E V S G E F F D Y K K Y Met G F Q K P S N P C L F L T

#K GTCGAGGAGCGCAA----------------------------------------AATCTATCCTCTCCCACTCACGATGAAGTCTCTGGTGAGTTCTTCGACTATAAGAAATATATGGGTTTCCAAAAGCCATCTAACCCATGTCTATTCCTAACC -40

V E E R K I Y P L P L T Met K S L V S S S T I R N I W V S K S H L T H V Y S *

#L GTCGAGGAGCGCAACAT-----GGAGTCAGCAAGTCGATGAGGCCTCGTACGAAAATCTATCCTCTCCCACTCACGATGAAGTCTCTGGTGAGTTCTTCGACTATAAGAAATATATGGGTTTCCAAAAGCCATCTAACCCATGTCTATTCCTAACC -5

V E E R N Met E S A S R *

#Q GTCGAGGAGCGCAACAT-ccatggagtcagCAAGTCGATGAGGCCTCGTACGAAAATCTATCCTCTCCCACTCACGATGAAGTCTCTGGTGAGTTCTTCGACTATAAGAAATATATGGGTTTCCAAAAGCCATCTAACCCATGTCTATTCCTAACC -1

V E E R N I H G V S K S Met R P R T K I Y P L P L T Met K S L V S S S T I R N I W V S K S H L T H V Y S *

ime4/mettl3

mettl14

fl(2)d

YT521-B

CG6422

Supplementary Figure 6. CRISPR-induced mutations in m6A pathway factors. Shown are genomic details of the five m6A factors subjected to CRISPR/Cas9-mediated mutagenesis. The locations of frame-shift indels are indicated above each locus, and the genomic alterations and predicted mutant proteins are shown below each locus.

y = 0.003791x R = 0.999756

0

2

4

6

8

10

12

14

16

0 500 1000 1500 2000 2500 3000 3500

Nor

mal

ized

abu

ndan

ce

Amount of nucleoside (fmol)

y = 0.032x R = 0.99958

0

2

4

6

8

10

12

14

16

50 100 150 200 250 300 350 400 450 500

Nor

mal

ized

abu

ndan

ce

Amount of nucleoside (pmol)

N6-methyladenosine (m6A) Adenosine (rA)

yw-F yw-F CG5933sk2-

homo-F

CG5933sk2-

homo-F

CG7818sk1-

homo-F

CG7818sk2-

homo-F

CG5933sk2-def-F

CG5933sk2-def-F

CG7818sk1-def-F

CG7818sk1-def-F

CG7818sk2-def-F

CG7818sk2-def-F

w1118 Nito

m6Am 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0.0

0.2

0.4

0.6

0.8

1.0

m6A

m /

10^5

nuc

leos

ides

N6,2'-O-dimethyladenosine (m6Am) - total RNA

A B

D

mettl3homozygous

mettl14homozygous

mettl3hemizygous

mettl14hemizygous

mettl14hemizygous

control control nitohomozygous

Adult femaletotal RNA

3rd instar larvae(both sexes)

total RNA

Supplementary Figure 7. Calibration curves for m6A quantification and m6Am analysis. Shown are standard calibration curves for detection of adenosine (A) and N6-methyladenos-ine (B) used for absolute quantifications. Inset on (B) shows the lower quantification range. (C) Modest reduction in m6A in nito homozygous mutant larvae compared to control w[1118]. The modest reduction might be due to maternal deposits and/or incomplete rRNA depletion by single dT selection. (D) Failure to detect m6Am in total RNA of control and m6A pathway mutants.

0.00

0.01

0.02

0.03

0.04

m6A

/100

ade

nosi

ne

m6A in pA+ RNA(1 dT selection)

w[1118]

nito[1/1]

3rd instar larvae

C

AdM_Ecl_30daysAdM_Ecl_5daysAdM_Ecl_1days

AdF_Ecl_30daysAdF_Ecl_5daysAdF_Ecl_1days

P15P9−10

P8P6P5

WPPL3_PS7−9L3_PS3−6L3_PS1−2

L3_12hrL2L1

em22−24hrem20−22hrem18−20hrem16−18hrem14−16hrem12−14hrem10−12hr

em8−10hrem6−8hrem4−6hrem2−4hrem0−2hr

0 30 60 90 120

AdM_Ecl_30daysAdM_Ecl_5daysAdM_Ecl_1days

AdF_Ecl_30daysAdF_Ecl_5daysAdF_Ecl_1days

P15P9−10

P8P6P5

WPPL3_PS7−9L3_PS3−6L3_PS1−2

L3_12hrL2L1

em22−24hrem20−22hrem18−20hrem16−18hrem14−16hrem12−14hrem10−12hr

em8−10hrem6−8hrem4−6hrem2−4hrem0−2hr

0 30 60 90 120

AdM_Ecl_30daysAdM_Ecl_5daysAdM_Ecl_1days

AdF_Ecl_30daysAdF_Ecl_5daysAdF_Ecl_1days

P15P9−10

P8P6P5

WPPL3_PS7−9L3_PS3−6L3_PS1−2

L3_12hrL2L1

em22−24hrem20−22hrem18−20hrem16−18hrem14−16hrem12−14hrem10−12hr

em8−10hrem6−8hrem4−6hrem2−4hrem0−2hr

0 30 60 90 120

AdM_Ecl_30daysAdM_Ecl_5daysAdM_Ecl_1days

AdF_Ecl_30daysAdF_Ecl_5daysAdF_Ecl_1days

P15P9−10

P8P6P5

WPPL3_PS7−9L3_PS3−6L3_PS1−2

L3_12hrL2L1

em22−24hrem20−22hrem18−20hrem16−18hrem14−16hrem12−14hrem10−12hr

em8−10hrem6−8hrem4−6hrem2−4hrem0−2hr

0 30 60 90 120

AdM_Ecl_30daysAdM_Ecl_5daysAdM_Ecl_1days

AdF_Ecl_30daysAdF_Ecl_5daysAdF_Ecl_1days

P15P9−10

P8P6P5

WPPL3_PS7−9L3_PS3−6L3_PS1−2

L3_12hrL2L1

em22−24hrem20−22hrem18−20hrem16−18hrem14−16hrem12−14hrem10−12hr

em8−10hrem6−8hrem4−6hrem2−4hrem0−2hr

0 30 60 90 120

AdM_Ecl_30daysAdM_Ecl_5daysAdM_Ecl_1days

AdF_Ecl_30daysAdF_Ecl_5daysAdF_Ecl_1days

P15P9−10

P8P6P5

WPPL3_PS7−9L3_PS3−6L3_PS1−2

L3_12hrL2L1

em22−24hrem20−22hrem18−20hrem16−18hrem14−16hrem12−14hrem10−12hr

em8−10hrem6−8hrem4−6hrem2−4hrem0−2hr

0 30 60 90 120

AdM_Ecl_30daysAdM_Ecl_5daysAdM_Ecl_1days

AdF_Ecl_30daysAdF_Ecl_5daysAdF_Ecl_1days

P15P9−10

P8P6P5

WPPL3_PS7−9L3_PS3−6L3_PS1−2

L3_12hrL2L1

em22−24hrem20−22hrem18−20hrem16−18hrem14−16hrem12−14hrem10−12hr

em8−10hrem6−8hrem4−6hrem2−4hrem0−2hr

0 30 60 90 120

spenito

YT521−B (CG12076)

CG6422

modENCODE DevelopmentalTimecourse libraries

Ime4 (CG5933)

CG7818

fl(2)d

virilizer

WPP_salivWPP_fat

L3_Wand_salivP8_fat

L3_Wand_imag_discL3_Wand_fat

L3_Wand_dig_sysA_4d_dig_sys

A_20d_dig_sysA_1d_dig_sys

L3_Wand_carcassA_4d_carcass

A_20d_carcassA_1d_carcass

A_MateM_4d_acc_glandA_MateM_4d_testis

A_VirF_4d_ovaryA_MateF_4d_ovary

A_VirF_20d_headA_MateM_4d_head

A_MateM_20d_headA_MateF_4d_head

A_MateF_20d_headA_VirF_4d_headA_VirF_1d_head

A_MateM_1d_headA_MateF_1d_head

P8_CNSL3_CNS

0 30 60 90 120

Ime4 (CG5933)

WPP_salivWPP_fat

L3_Wand_salivP8_fat

L3_Wand_imag_discL3_Wand_fat

L3_Wand_dig_sysA_4d_dig_sys

A_20d_dig_sysA_1d_dig_sys

L3_Wand_carcassA_4d_carcass

A_20d_carcassA_1d_carcass

A_MateM_4d_acc_glandA_MateM_4d_testis

A_VirF_4d_ovaryA_MateF_4d_ovary

A_VirF_20d_headA_MateM_4d_head

A_MateM_20d_headA_MateF_4d_head

A_MateF_20d_headA_VirF_4d_headA_VirF_1d_head

A_MateM_1d_headA_MateF_1d_head

P8_CNSL3_CNS

0 30 60 90 120

CG7818

WPP_salivWPP_fat

L3_Wand_salivP8_fat

L3_Wand_imag_discL3_Wand_fat

L3_Wand_dig_sysA_4d_dig_sys

A_20d_dig_sysA_1d_dig_sys

L3_Wand_carcassA_4d_carcass

A_20d_carcassA_1d_carcass

A_MateM_4d_acc_glandA_MateM_4d_testis

A_VirF_4d_ovaryA_MateF_4d_ovary

A_VirF_20d_headA_MateM_4d_head

A_MateM_20d_headA_MateF_4d_head

A_MateF_20d_headA_VirF_4d_headA_VirF_1d_head

A_MateM_1d_headA_MateF_1d_head

P8_CNSL3_CNS

0 30 60 90 120

fl(2)d

WPP_salivWPP_fat

L3_Wand_salivP8_fat

L3_Wand_imag_discL3_Wand_fat

L3_Wand_dig_sysA_4d_dig_sys

A_20d_dig_sysA_1d_dig_sys

L3_Wand_carcassA_4d_carcass

A_20d_carcassA_1d_carcass

A_MateM_4d_acc_glandA_MateM_4d_testis

A_VirF_4d_ovaryA_MateF_4d_ovary

A_VirF_20d_headA_MateM_4d_head

A_MateM_20d_headA_MateF_4d_head

A_MateF_20d_headA_VirF_4d_headA_VirF_1d_head

A_MateM_1d_headA_MateF_1d_head

P8_CNSL3_CNS

0 30 60 90 120

virilizer

51-10026-50

11-254-101-3ex

pres

sion

sca

le

RPKM101-1000

WPP_salivWPP_fat

L3_Wand_salivP8_fat

L3_Wand_imag_discL3_Wand_fat

L3_Wand_dig_sysA_4d_dig_sys

A_20d_dig_sysA_1d_dig_sys

L3_Wand_carcassA_4d_carcass

A_20d_carcassA_1d_carcass

A_MateM_4d_acc_glandA_MateM_4d_testis

A_VirF_4d_ovaryA_MateF_4d_ovary

A_VirF_20d_headA_MateM_4d_head

A_MateM_20d_headA_MateF_4d_head

A_MateF_20d_headA_VirF_4d_headA_VirF_1d_head

A_MateM_1d_headA_MateF_1d_head

P8_CNSL3_CNS

0 30 60 90 120

WPP_salivWPP_fat

L3_Wand_salivP8_fat

L3_Wand_imag_discL3_Wand_fat

L3_Wand_dig_sysA_4d_dig_sys

A_20d_dig_sysA_1d_dig_sys

L3_Wand_carcassA_4d_carcass

A_20d_carcassA_1d_carcass

A_MateM_4d_acc_glandA_MateM_4d_testis

A_VirF_4d_ovaryA_MateF_4d_ovary

A_VirF_20d_headA_MateM_4d_head

A_MateM_20d_headA_MateF_4d_head

A_MateF_20d_headA_VirF_4d_headA_VirF_1d_head

A_MateM_1d_headA_MateF_1d_head

P8_CNSL3_CNS

0 30 60 90 120

WPP_salivWPP_fat

L3_Wand_salivP8_fat

L3_Wand_imag_discL3_Wand_fat

L3_Wand_dig_sysA_4d_dig_sys

A_20d_dig_sysA_1d_dig_sys

L3_Wand_carcassA_4d_carcass

A_20d_carcassA_1d_carcass

A_MateM_4d_acc_glandA_MateM_4d_testis

A_VirF_4d_ovaryA_MateF_4d_ovary

A_VirF_20d_headA_MateM_4d_head

A_MateM_20d_headA_MateF_4d_head

A_MateF_20d_headA_VirF_4d_headA_VirF_1d_head

A_MateM_1d_headA_MateF_1d_head

P8_CNSL3_CNS

0 30 60 90 120

modENCODETissue libraries

spenito

YT521−B (CG12076)

CG6422

Supplementary Figure 8. Expression of m6A-related factors across modENCODE data. Shown are the RPKM measurements of the indicated factors across diverse developmental time-course datasets (left column) or dissected tissue datasets (right column) produced by the modENCODE project. Note that all factors exhibit maximal or preferential expression in neural libraries (e.g., larval or pupal CNS, or adult heads), and that gonads (ovaries or testes) represent another location of elevated expression of m6A-related factors.

yw

ime4

[SK2]/

Df

mettl14

[SK1]/

Df

YTH521-B

[NP3]/

Df

CG6422

[NP3]/

Df0

20

40

60

80

100

120

7.95E-05

ns

0.000210.00015

% m

ales

with

hel

d-ou

t win

gs

yw

ime4

[SK2]/

Df

mettl14

[SK1]/

Df

YTH521-B

[NP3]/

Df

CG6422

[NP3]/

Df0

20

40

60

80

100

120

1.49E-13

7.25E-12

0.75

3.54E-17

% m

ales

clim

bing

7cm

in 1

0s

A B

Supplementary Figure 9. Defective negative geotaxis and held-out wings in m6A-pathway mutant males. (A) Negative geotaxis assay. 10 flies were placed in an empty vial and tapped to the bottom, and their ability to climb was quantified. Five independent cohorts of flies per genotype were assays, and the assay was done in triplicate for each group of flies. Nearly all control (yw) flies cross the 7cm mark within 10 seconds; indeed, nearly all of these reached this mark in <5 seconds. Most ime4 hemizygotes stayed at the bottom of the vial, and a minority slowly climb to the designated height. mettl14 and YT521-B hemizygous mutants also display strongly reduced negative geotaxis, whereas CG6422 hemizygotes were normal. (B) Quantification of held-out wings in the indicated genotypes. This wing posture defect is roughly correlated with the presence of locomotor defects quantified in other assays.

mettl14[SK1]/Df

ime4[SK2]/Df

yw

yw

ime4[SK2]/Df

mettl14[SK1]/Df

YT521-B[NP3]/Df

0

1

2

3

4

5

6

# eg

g ch

ambe

rs p

er o

vario

le

1

21

2

3

1

2

1

2basalstalk

12

31

23 4

12

3

VAS

A H

ts D

AP

I

G Hn=90 n=97 n=128 n=114

1 2

3

1 2 3

4

5

germariumSt 1

St 2 St 4 St 6

St 8

St 9

12

3

St 1

St 2

St 6

St 9

empty

YT521-B[NP3]/Dfmettl14[SK1]/Df

YT521-B[NP3]/Df

A C E

B D F12

3

St 1St 4

St 6

St 9

St 1 St 2 St 6

St 9

St 1

St 4

St 2

St 9

Supplementary Figure 10. Oogenesis phenotypes of m6A pathway mutants (A–F) Ovaries were stained with anti-Hts (red) and anti-Vasa (green) antibodies. Germarium structures are marked with dotted outlines, and the numbers of egg chambers in selected individual ovarioles are labeled with white numbers. The stages of selected egg chambers are labeled in yellow. (A) Control yw genotype illustrates the normal progression of an ovariole with a germarium followed by a string of egg chambers of various stages, in this case five egg chambers going up to stage 9. (B) ime4 hemizygous ovarioles showing abbreviated sets of egg chambers. (C-D) Examples of mettl14 hemizygous ovarioles, illustrating either abbreviated strings of egg chambers (C) or an ovariole that contains a relatively mature stage 9 egg chamber but misses some earlier stages (D). (E-F) Examples of YT521-B hemizygous ovarioles, which either show mild loss of egg chambers (E) or a rarer class of empty ovariole lacking egg chambers (F). scale bars, 50 µm. (G) Quantification of egg-cham-ber number shows that different m6A pathway mutant ovaries exhibit fewer than in yw control. (H) Distribution of egg chamber stages shows a skew towards early stages in m6A pathway mutants relative to yw control.

yw

ime4[SK2]/Df

mettl14[SK1]/Df

YT521-B[NP3]/Df

0

20

40

60

80

100

no egg chamberst 4st 6st 8

n=102 n=100 n=196 n=156

st 9compound chamber

mos

t mat

ure

stag

e pr

esen

t(%

of o

vario

les)

0 50 100 150 200

yw

*** ns

(n=327)

(n=105)

(n=338)

(n=104)

(n=58)

(n=460)

***

*** ns

ns

***

CG6422[NP2/NP2]

YT521-B[NP3]/Df(3L)ED208

YT521-B[NP3/NP3]

mettl14[SK1]/Df(2L)BSC111

ime4[SK2]/Df(3R)Exel6197

Supplementary Figure 11. Additional analysis of female survival in m6A pathway mutants. (Top) This graph summarizes the female survival as a percentage of male siblings in various m6A pathway mutants. Female lethality is not observed, in contrast to genetic sensitization experi-ments involving Sxl heterozygosity. (Bottom). Genetic interaction tests of Sxl, da and CG6422. Maternal heterozygosity of da combined with paternal Sxl heterozygosity results in ~50% female lethality. This was not modified by two of the CG6422 alleles, but inclusion of CG6422[NP3] dominantly enhanced female lethality.

0 20 40 60 80 100

CG6422[NP3]/+

CG6422[NP2]/+

CG6422[NP1]/+

da[3]/+ (n=937)

(n=1105)

ns

**

Maternal genotype X Sxl[7BO]/Y Fathers

(n=691)

(n=818)ns

% Female viability (relative to male progeny)

n=# male progeny

da[3]/+;da[3]/+;

da[3]/+;

Maternal genotype X yw Fathers

% Female viability (relative to male progeny)

n=# male progeny

stj fl(2)d CG13339

0

54.495

0

9.874

0

70.225

0

9.075

0

53.079

0

8.439

0

28.551

0

6.605

0

31.06

0

4.186

0

13.958

0

2.995

9,707,000 bp 9,708,000 bp 9,709,000 bp 9,710,000 bp 9,711,000 bp 9,712,000 bp

6,373 bp

Female lethal 2D - fl(2)d

T1 miCLIP

T2 miCLIP

T3 miCLIP

T4 miCLIP

T5 miCLIP

T6 miCLIP

T1 mRNA

T2 mRNA

T3 mRNA

T4 mRNA

T5 mRNA

T6 mRNA

T1 CIMS

T2 CIMS

T3 CIMS

T4 CIMS

T5 CIMS

T6 CIMS

Supplementary Figure 12. Summary of mRNA-seq, miCLIP and CIMS calls at fl(2)d. Along with Sxl (Figure 7C-D), fl(2)d is one of the top loci in the genome in terms of intronic CIMS calls (see also Supplementary Table S6). (Top) Summary of miCLIP, mRNA-seq and CIMS tracks at fl(2)d. A prominent set of CIMS calls is observed in its first intron, in addition to a 5’ UTR peak. (Bottom) Analysis of RNA-seq and miCLIP expression (left Y-axis), and exonic/intron CIMS sites (right Y-axis) across embryogenesis. These summaries emphasize that miCLIP and CIMS calls at f(l)2d are found specifically in datasets following activation of zygotic transcription.

0

50

100

150

T1 T2 T3 T4 T5 T6

FPK

M m

RN

A o

r miC

LIP exonic or intronic C

IMS

0

2

4

6miCLIP

mRNA

exonic CIMS

intronic CIMSfl(2)d

100

70100

130

55

35

70

100

130

55

35

HA-IME4

myc

-GFP

GFP

-ME

TTL1

4

GFP

-Nito

GFP

-fl(2

)d

70

55

7055

HA-METTL14

myc

-GFP

GFP

-IME

4

GFP

-Nito

GFP

-fl(2

)d

METTL14

Fl(2)d

Input,anti-HA

Inputanti-GFP

IME4Fl(2)d

Figure 2E Figure 2F

Figure 2E Figure 2F

100

Figure 2E Figure 2F

HA-IP,anti-GFP

HA-IME4

myc

-GFP

GFP

-ME

TTL1

4

GFP

-Nito

GFP

-fl(2

)d

HA-METTL14

myc

-GFP

GFP

-IME

4

GFP

-Nito

GFP

-fl(2

)d

HA-IME4

myc

-GFP

GFP

-ME

TTL1

4

GFP

-Nito

GFP

-fl(2

)d

HA-METTL14

myc

-GFP

GFP

-IME

4

GFP

-Nito

GFP

-fl(2

)d

HA-IME4

myc

-GFP

GFP

-ME

TTL1

4

GFP

-Nito

GFP

-fl(2

)d

HA-METTL14

myc

-GFP

GFP

-IME

4

GFP

-Nito

GFP

-fl(2

)d

Input,anti-HA

Figure 2E Figure 2F

GFP-IP:anti-GFP

GFP-Nito

HA-Fl(2

)d

GFP-Nito

+ HA-F

l(2)d

myc-G

FP + HA-F

l(2)d

Input,anti-HA

GFP-IP,anti-HA

10075

10075

75100150

5037

Figure 2G

GFP-IP: anti-GFPGFP-IP: anti-HA Input: anti-HA

GFP-Nito

+HA-M

ettl14

GFP+HA-M

ettl14

GFP-Nito

+HA-Im

e4

GFP+HA-Im

e4

GFP-Nito

+HA-M

ettl14

GFP+HA-M

ettl14

GFP-Nito

+HA-Im

e4

GFP+HA-Im

e4

GFP-Nito

+HA-M

ettl14

GFP+HA-M

ettl14

GFP-Nito

+HA-Im

e4

GFP+HA-Im

e4

250

15010075

50

37

2520

1510

250

15010075

50

37

25

20

1510

250

15010075

50

37

2520

1510

Figure 2H

70100

130

250

55

3525

yw ime4[SK2]/TM6C

ime4[SK2]/Df

ime4[SK2/SK2]

mettl14[SK1]/CyO

mettl14[SK1/SK1]

70100130

250

55

3525

yw ime4[SK2]/TM6C

ime4[SK2]/Df

ime4[SK2/SK2]

mettl14[SK1]/CyO

mettl14[SK1/SK1]

anti-IME4 anti-tubulin

Figure 3B

Supplementary Figure 13. Uncropped Western blots for data presented in the main figures.

HA-IP,anti-HA