Magdalena Natalia Wojtas, Radha Raman Pandey, Mateusz ... · Sachidanandam, and Ramesh S. Pillai....

Molecular Cell, Volume 68

Supplemental Information

Regulation of m6A Transcripts by the 3ʹ/5ʹ RNA

Helicase YTHDC2 Is Essential for a Successful

Meiotic Program in the Mammalian Germline

Magdalena Natalia Wojtas, Radha Raman Pandey, Mateusz Mendel, David Homolka, RaviSachidanandam, and Ramesh S. Pillai

SUPPLEMENTAL FIGURE LEGENDS

Figure S1. Production of recombinant YTHDC2 protein. Related to Figure 1 and 5.

(A) Protein sequence alignment of YTH domains from human YTHDC1 (hDC1) and YTHDC2 (hDC2).

Secondary structure features encoded by the protein sequence, as present in crystal structure of YTH

domain of YTHDC1 (PDB: 4R3H) and solution structure of YTH domain of YTHDC2 (PDB: 2YU6), are

indicated above. The YTH domain is made up of a mix of helices and sheets. (B) Gel-filtration elution

profile of the 6xHis-SUMO-tagged YTH domain of hYTHDC2 that was used for RNA Bind-N-Seq

experiment. The protein elutes as a single peak at a volume consistent with its predicted molecular weight.

See also Figure 1D. (C) Nucleotide frequencies plotted at individual positions along the 21 nt RNA reads

that are enriched by the 6xHis-SUMO-tagged YTH domain or present in the beads alone control. Position

11 corresponds to the central m6A moiety that is same for all sequences, so not plotted. See also Figure 1E.

(D) Purification of full-length untagged hYTHDC2. An Instant Blue-stained SDS-PAGE with fractions

from a gel-filtration column purification is shown. Fraction volumes correspond to the expected molecular

weight of the protein. See biochemical activities of this protein in Figure 5. (E) SDS-PAGE gel of gel-

filtration fractions of hYTHDC2YTH protein. (F) Gel-filtration chromatography profile for indicated

proteins. Note that there is no dramatic difference in the elution profile of the ATPase mutant (E332Q or

D/Q) when compared to the wildtype protein. See also Figure 5. (G) Mass spectrometry analysis of

mYTHDC2 immunopurification from adult mouse testes. Experiments were carried out in triplicates for

the following conditions: anti-mYTHDC2 immunopurification with or without RNase-treatment, and

negative control (beads bound with normal mouse IgG). The reported interaction partner MEIOC is detected

among the top ten candidates. XRN1 was present, but only in one of the samples. Total Spectrum Count is

shown. Protein threshold: 99%; min. peptides: 1; Peptide threshold: 90%. Presented protein hits are the top

10 with the highest number of peptides in the mYTHDC2 immunopurification samples and with no more

than 5 peptides in the negative control (normal mouse IgG) samples. (H) Mass spectrometry analysis of

five replicates of 3xFLAG-HA-hYTHDC2 immunopurification from transfected HeLa cells. 3xFLAG-HA-

GFP was used as negative control. XRN1 is the top interacting factor. Tandem-affinity purification was

performed (STAR METHODS). Exclusive Unique Peptide Count shown. Protein threshold: 1% FDR; min.

peptides: 2; Peptide threshold: 0.1% FDR. Presented protein hits are the top 10 with the highest number of

peptides in the 3xFLAG-HA-hYTHDC2 immunopurification samples and with no more than 5 peptides in

the negative control (3xFLAG-HA-GFP) samples. See also Figure 5E-F. (I) Immunoprecipitation of

endogenous YTHDC2 from adult mouse testes lysates. Detection of co-precipitated endogenous XRN1.

Replicate of experiment shown in Figure 5D.

Figure S2. Generation of Ythdc2 knock-out mice. Related to Figure 2 and 3.

(A) Multiple-tissue Western analysis of mYTHDC2 using adult mouse tissue lysates. This blot was probed

with the anti-mYTHDC2 rabbit polyclonal antibodies prepared for this study. (B) Exon model of mouse

Ythdc2 gene. Exon 7 encodes for the ATPase motif DEVH found in the RNA helicase domain. Two guide

RNAs (gRNAs; only gene-specific sequences are shown) were made that cleave around exon 7 to delete it

(line #2). One of the gRNA was also used for knocking-in a triple-stop cassette (using a ssDNA repair

template) within the exon 7 (line #1). Both result in truncation of coding sequence and probably results in

decay of the mutant transcript by nonsense-mediated decay, causing loss of the protein product. See Figure

2C. (C) Agarose gel showing PCR products obtained with genomic DNA from mouse ear-punches.

Expected band sizes of the wildtype and mutant alleles are indicated. PCR products were sequenced to

confirm identity and partial sequences are shown in the alignment in panel B to indicate the mutations

made. (D) Histological analysis of ovaries from adult (P60) Ythdc2 knock-out animals. Note the complete

absence of any follicles in different biological replicates from both line #1 and 2. It is likely that apopotosis

of arrested germ cells results in their complete removal in these animals. See Figure 2E, where we present

one of the adult mutant female with ovaries containing immature follicles. (E) Histological analysis of

ovaries from newborn (P0) animals of the indicated genotypes. Scale bars are indicated. See also Figure

2G. (F) Comparison of read annotations in biological duplicate ovarian P0 total RNAseq datasets from

Ythdc2-/- and Ythdc2+/-. Note that there is an increased representation of gene intronic sequences in all

libraries. (G) Expression of individual genes was compared between Ythdc2-/- and Ythdc2+/-. The density

plot is shown and the genes with significantly different expression (adjusted p-value <0.1) are shown as red

dots. (H) Top ten enriched GO terms in the Biological Process ontology are shown for the genes

downregulated in P0 ovarian RNAseq dataset of Ythdc2-/-. The enriched categories were identified using

ENRICHR. Note that many of the downregulated genes are active piRNA pathway components, but our

examination of repeat elements did not reveal any upregulation of these in the mutant. This is consistent

with the absence of any significant role for the piRNA pathway in the female germline. Given that the

sampling window (P0) is much later than the meiotic arrest in the female germline (which is embryonic),

much of the changes recorded here could be a consequence of the arrested state.

Figure S3. Analyses of spermatogenesis in Ythdc2 knock-out mice. Related to Figure 3.

(A) Global histological view of adult (P60) mouse testes sections from animals of indicated genotypes.

Note the narrow, empty tubules in the Ythdc2-/- mutant. Age of donor animals is P60 (60 days after birth).

(B) Histology of adult mouse testes showing narrow tubules in the Ythdc2-/- mutant. Scale bars are indicated.

(C) TUNEL assay with adult testes sections from indicated genotypes. Note the increased signal from the

Ythdc2-/- mutant, indicative of increased cell death. See Figure 3. (D) Histological analyses of P12 testes.

Note the presence of germ cells in the zygotene (Zyg) stage of meiosis in the control Ythdc2+/- testes, but

lacking in the Ythdc2-/- mutant. Spermatogonia (Spg) are seen in both animals. The Ythdc2-/- mutant has

increased number of dead apoptotic (Apo) cells and cells that have entered an aberrant metaphase (Met)

state. See also Figure 3G. (E) Immunofluorescence analysis of P12 testes from animals of indicated

genotypes. PLZF-marked undifferentiated spermatogonia are found in both control and mutant testes. (F)

Histological analyses of P8 testes from animals of indicated genotypes. The cellularity is homogeneous in

P8 animals, but start to show molecular defects as presented in Figure 4J.

Figure S4. Analyses of testicular transcriptome from P12 Ythdc2 knock-out mice. Related to Figure

3.

(A) Comparison of read annotation in total testicular RNAseq of Ythdc2+/- and Ythdc2-/- P12 (12 days old)

animals. The error bars correspond to minimal and maximal values of the biological replicas. See also

Figure 3. (B) Transcripts downregulated in total testicular RNAseq dataset from P12 Ythdc2-/- (compared

to Ythdc2+/-) were identified and a heatmap of their expression in individual spermatogenic populations

plotted. SSC, spermatogonial stem cells; SC, pachytene spermatocytes; RS, round spermatids. Transcripts

normally enriched in meiotic pachytene spermatocytes and post-meiotic round spermatids are

downregulated in the P12 RNAseq dataset from the Ythdc2-/- mutant. This is consistent with the difference

in cellularity at this stage, given that some germ cells in the Ythdc2+/- control animals proceed to the

zygotene stage of meiosis, and these are lacking in the mutant. See Figure S3D. (C) Top ten enriched GO

terms in the Biological Process ontology are shown for the genes upregulated in P12 testicular RNAseq

dataset of the Ythdc2-/- mutant. The enriched categories were identified using ENRICHR. See also Figure

3I. (D) Top ten enriched GO terms in the Biological Process ontology are shown for the genes

downregulated in P12 testicular RNAseq dataset of Ythdc2-/-.

Figure S5. Analyses of the mouse male germline m6A transcriptome and its regulation by YTHDC2.

Related to Figure 4.

(A) The sequence motif identified in the 21nt sequences centred on the summits of m6A coverage peaks.

The motif was searched using MEME - Motif discovery tool. See also Figure 4B. (B) Normalized read

coverage is plotted along the transcripts divided into 100 parts (from 5' to 3' end). Top 500 m6A-IP-enriched

genes were identified for every stage during spermatogenesis (from libraries prepared with P8, P12, P20

mouse testes) and the lists merged to yield the final list of 643 genes. The mean coverage was calculated

from the parts of corresponding transcripts and plotted for individual samples. See Figure 4. (C) Normalized

read coverage is plotted along the first, penultimate (before last) and last exon, which were divided into 100

parts (from 5' to 3' end). Data from top 500 m6A-IP enriched genes are shown. See also Figure 4E. (D)

Heatmap of expression of 4250 genes which were found to be significantly enriched in m6A-IP libraries in

all three stages tested (P8, P12, P20). Data for individual spermatogenic populations comes from a public

dataset (European Nucleotide Archive - Study: PRJEB15333). SSC, spermatogonial stem cells; SC,

pachytene spermatocytes; RS, round spermatids. (E) Comparison of read annotation in P8 total testicular

RNAseq datasets from Ythdc2+/+, Ythdc2+/-, Ythdc2-/-. The error bars correspond to minimal and maximal

values of the biological replicas. (F) Expression of individual genes was compared between Ythdc2-/-,

Ythdc2+/- and Ythdc2+/+. The density plots are shown and the genes with significantly different expression

(adjusted p-value <0.1) are shown as red dots. (G) Heatmap of expression of the genes which were found

to be significantly dysregulated in any of the compared genotypes.

SUPPLEMENTAL TABLE LEGENDS

Supplemental Table 1. List of all deep-sequencing libraries created in this study. Related to STAR

Methods and Figure 3 and 4.

Data is available from GEO under accession no. GSE102346.

Supplemental Table 3. Genes with significantly different expression in P8 testes of Ythdc2-/- vs

Ythdc2+/- and Ythdc2-/- vs Ythdc2+/+. Related to STAR Methods and Figure 4.

Supplemental Table 5. DNA primers and RNA oligonucleotides used in this study. Related to STAR

Methods.

D

Figure-S1

E

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A B

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S75 10/300 GLSUMO-hYTHDC2 YTH domain

mAU

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IPbeads

hDC2hDC1

β1

α1 β2 α2 β3 β4 β5

=

β6 β7 α3 β8 β9 β10 α4 η1

2YU6

hDC2hDC1

2YU6

4R3H

4R3H

RYFIMKSSNLRNLEISQQKGIWSTTPSNERKLNRAFWESSIVYLVFSVQGSGHFQGFSRMSSEIGRRFFLIKSNNHENVSLAKAKGVWSTLPVNEKKLNLAFRSARSVILIFSVRESGKFQGFARLSSESHH

EKSQD.W......GSAGLGGVFKVEWIRKESLPFQFAHHLLNPWNDNKKVQISRDGQELEPLVGEQLLQLWE GGSPIHWVLPAGMSAKMLGGVFKIDWICRRELPFTKSAHLTNPWNEHKPVKIGRDGQEIELECGTQLCLLFP

356

1289

C

hYTHDC2 WThYTHDC2 D/QhYTHDC2 ∆YTH (1-1219aa)

0102030405060708090

0 5 10 15 20 25

S200 10/300 GL

mAU

ml

Instant Blue stain

hYTHDC2S200 gel-filtration column elutions

Input

kDa

180

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120

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40

35

hYTHDC2 ∆YTH

Instant Blue stain

kDa

180

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120

55

40

S200 gel-filtration elutions

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No Protein name Gene symbol Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 31 Cluster of E3 ubiquitin-protein ligase MYCBP2 Mycbp2 43 19 26 39 34 23 0 0 02 Citron Rho-interacting kinase Cit 24 13 10 31 20 13 0 0 03 Acrosomal protein KIAA1210 Kiaa1210 28 13 26 30 25 24 1 0 24 Protein scribble homolog Scrib 24 18 23 30 24 26 0 0 05 Cluster of Echinoderm microtubule-associated protein-like 4 Eml4 24 18 8 22 17 11 0 0 06 Rho guanine nucleotide ex change factor 12 Arhgef12 18 11 11 21 18 17 0 0 07 Meiosis-specific coiled-coil domain-containing protein MEIOC Meioc 10 3 6 20 7 12 0 0 08 Zinc finger protein 541 Znf541 18 11 9 16 15 13 0 0 09 Cluster of BTB/POZ domain-containing protein KCTD19 Kctd19 19 5 4 16 11 7 0 0 0

10 Cluster of Zinc finger CCCH domain-containing protein 14 Zc3h14 1 3 2 12 5 4 0 0 0

No Protein name Gene symbol Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 3 Rep 1 Rep 2 Rep 31 5'-3' ex oribonuclease 1 Xrn1 0 0 0 1 0 0 0 0 0

-RNase +RNase CTRL

Total Spectrum Count-RNase +RNase CTRL

Total Spectrum Count

No Identified Proteins

Gene symbol

Rep 1Rep 2Rep 3Rep 4Rep 5Rep 1Rep 21 Probable ATP-dependent RNA helicase YTHDC2 YTHDC2 136 128 127 100 129 0 02 5'-3' ex oribonuclease 1 XRN1 23 21 25 2 30 0 03 Protein arginine N-methy ltransferase 5 PRMT5 17 29 22 12 25 0 04 Elongation factor 2 EEF2 16 13 11 8 14 3 35 RNA-binding protein 10 RBM10 13 21 10 4 14 0 06 Eukary otic translation initiation factor 4B EIF4B 11 14 10 4 12 0 07 Cluster of Heterogeneous nuclear ribonucleoprotein H HNRNPH1 12 11 9 8 11 0 08 40S ribosomal protein S3 RPS3 8 7 7 6 6 5 09 Ubiquitin carbox y l-terminal hy drolase 7 USP7 8 7 4 0 10 0 0

10 X-ray repair cross-complementing protein 6 XRCC6 8 9 5 3 3 0 0

HA-YTHDC2 WT HA-GFPExclusive Unique Peptide Count

G

H

I

XRN1

YTHDC2

WesternAdult mouse testes

Input (

2%)

-RNase+R

NasemYTHDC2 IP

Beads

IgG

Gene symbol

C

Figure-S2

TCTCTTCCTTGC ATATACAGTACGACTATCCATTATCTTCTTTAA S S L Q D E V H E R D R F S D F L L T ACGTCCTACTTCACGTACTT (gRNA) CACTATATATCATGCTGATA (gRNA)5’TCTCTTCCTTGCAGGATGAAGTGCATGAAAGGGATCGATTCAGTGATTTTTTGCTTACTA-/64bps/-AACCTCTTCATAAGATATTTTGGAAGTTGTCCAGTGATATATAGTACGACTATCCATTATC3’ |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||3’AGAGAAGGAACGTCCTACTTCACGTACTTTCCCTAGCTAAGTCACTAAAAAACGAATGAT-/64bps/-TTGGAGAAGTATTCTATAAAACCTTCAACAGGTCACTATATATCATGCTGATAGGTAATAG5’

(ssDNA)54 nt +AACCTCTTCATAAGATATTTTGGAAGTTGTCCAatgtaaatagatgaGTGATATATAGTA +67 nt

Line#1 genotyping PCR sequencing result AACCTCTTCATAAGATATTTTGGAAGTTGTCCAatgtaaatagatgaGTGATAT+ 83 nt random insertionExon 7 translation (WT) N L F I R Y F G S C P V I Y SExon 7 translation (Line#1) N L F I R Y F G S C P M *

PAM PAM

mm10: Chr18-Ythdc2ATPase motif: DEVH

152 nt deletion of genomic sequence + 4 nt random insertion

Line #2 has a large deletion that removes all of Exon 7 with the ATPase motif

Line #1 has an insertion of a TRIPLE STOP cassette into the Exon 7

1% Agarose gelGenotyping PCR

bp

100

500400300200

100

500400300200

Line #1 knock-in

Line #2 Deletion

WT

+/+ +/- +/-+/+ +/+ +/+ +/+ +/+Marker

Marker

bp

AAdult mouse tissues

Testis

Western; 8% SDS-PAGEAnti-mYTHDC2

TUBULIN

mYTHDC2

LungSpleen

KidneyBrain

170

kDa

55

B

D

P0 ovary

Ythdc2+/- Ythdc2-/-

20 µm 20 µm

E

Adult ovary (P60)Four more biological replicates of adult ovaries.

The animal ID and KO line # are indicated

Ythdc2-/-

50 µm

Animal #52629 (8773)-line #2

Animal #42529 (8772)-line #2

Animal #32680 (8771)-line #1

Animal #22233 (8264)-line #2

gene

exon

sens

e

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ense

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intro

n sen

se

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sens

e

repe

at se

nse

repe

at an

tisen

se

none

miRN

A se

nse

miRN

A an

tisen

se

% of

read

s

0

5

10

15

20

25

30

Ythdc2+/-

Ythdc2-/-

P0 ovary

F G

0 5 10 15

−2−1

01

2

Mean of normalized counts (log2)

Fold

chan

ge (lo

g2)

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Gene expressionP0 ovary: Ythdc2-/- vs Ythdc2+/-

23 upregulated genes

74 downregulated genes

● adjusted p-value < 0.1

Ythdc2

Ythdc2 -/- downregulated genes: enriched GO terms in the Biological Process ontologyTerm Overlap P-value Adjusted P-value Z-score Combined Score GenesDNA methylation involved in gamete generation (GO:0043046) 8/17 5.63E-16 5.66E-14 -3.01 91.84 PIWIL2;TDRD1;ASZ1;TDRD9;MAEL;MOV10L1;TDRD5;FKBP6piRNA metabolic process (GO:0034587) 8/15 1.50E-16 3.01E-14 -2.83 88.23 PIWIL2;TDRD1;EXD1;ASZ1;TDRD9;MAEL;MOV10L1;FKBP6spermatogenesis (GO:0007283) 11/136 2.77E-12 1.86E-10 -3.14 70.35 JAG2;TDRD1;NLRP14;PIWIL2;SHCBP1L;KIT;ASZ1;TDRD9;MAEL;MOV10L1;FKBP6gene silencing by RNA (GO:0031047) 5/12 4.69E-10 2.36E-08 -3.03 53.15 TDRD1;PIWIL2;EXD1;MAEL;FKBP6male meiotic nuclear division (GO:0007140) 3/16 2.63E-05 1.06E-03 -2.58 17.68 ASZ1;TDRD9;MAELT cell differentiation (GO:0030217) 3/20 5.30E-05 1.77E-03 -2.50 15.81 JAG2;CHD7;KITgerm cell development (GO:0007281) 2/11 7.27E-04 2.09E-02 -2.58 9.99 TDRD1;MOV10L1glutamate receptor signaling pathway (GO:0007215) 2/13 1.03E-03 2.58E-02 -2.29 8.37 GRID2;GRIK3heart morphogenesis (GO:0003007) 2/19 2.22E-03 4.46E-02 -2.42 7.52 TH;CHD7cell cycle (GO:0007049) 2/15 1.37E-03 3.07E-02 -1.90 6.61 JAG2;CCNO

H

Figure-S3

200 µm

A

Adult testes (P60)

Ythdc2+/- Ythdc2-/-

50 µm

Ythdc2+/- Ythdc2-/-

Adult testes (P60)

TUNE

L Ass

ay

C

D

20 µm

20 µm

20 µm 20 µm

10 µm10 µm

Ythdc2+/- Ythdc2-/-

P8 testes

F

Figure-S3

Ythdc2+/- Ythdc2-/-

PLZF

E

P12 Testes

10 µm

50µm

Ythdc2+/- Ythdc2-/-B

20 µm20 µm

P12 testes

Ythdc2+/- Ythdc2-/-

10 µm

Met

Apo

10 µm

Zyg

Spg

Adult testes (P60)

Figure-S4

AYthdc2 -/-Ythdc2 +/-

P12 testes

SSC

_1

SSC

_2

SC_1

SC_2

RS_

1

RS_

2

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

Gene expression in spermatogenic populations

B Ythdc2 -/- downregulated genes

Term Overlap P-value Adjusted P-value Z-score Combined Score Genesmitochondrial electron transport, cytochrome c to oxygen (GO:0006123) 2/20 2.59E-04 1.68E-02 -2.68 10.96 COX8A;COX7Bamino acid transport (GO:0006865) 2/32 6.70E-04 2.18E-02 -2.53 9.70 SLC38A1;SLC7A11protein deubiquitination (GO:0016579) 2/239 3.30E-02 6.32E-02 -3.27 9.03 CCNA2;ATXN3misfolded or incompletely synthesized protein catabolic process (GO:0006515) 1/8 9.56E-03 4.80E-02 -2.79 8.46 ATXN3glucocorticoid biosynthetic process (GO:0006704) 1/7 8.37E-03 4.80E-02 -2.76 8.37 CYP17A1G2/M transition of mitotic cell cycle (GO:0000086) 2/126 9.93E-03 4.80E-02 -2.69 8.18 CCNA2;PRKAR2Brespiratory burst (GO:0045730) 1/10 1.19E-02 4.80E-02 -2.55 7.74 CD52histone phosphorylation (GO:0016572) 1/8 9.56E-03 4.80E-02 -2.50 7.59 CCNA2negative regulation of cAMP-dependent protein kinase activity (GO:2000480) 1/8 9.56E-03 4.80E-02 -2.37 7.19 PRKAR2Bprogesterone metabolic process (GO:0042448) 1/7 8.37E-03 4.80E-02 -2.15 6.53 CYP17A1

CYthdc2 -/- upregulated genes: enriched GO terms in the Biological Process ontology

Term Overlap P-value Adjusted P-value Z-score Combined Score Genessynaptonemal complex assembly (GO:0007130) 7/11 6.45E-13 1.51E-10 -2.35 53.21 SYCE3;SYCE2;STAG3;SYCP2;SYCP1;TEX12;HORMAD1spermatogenesis (GO:0007283) 9/136 1.67E-06 1.30E-04 -3.14 28.08 SYCE3;TSGA10;SYCP1;SPO11;CCDC36;DMC1;TESK2;HSF2BP;HORMAD1reciprocal meiotic recombination (GO:0007131) 5/24 1.23E-06 1.30E-04 -2.49 22.29 SYCE3;SYCP1;SPO11;MSH4;DMC1meiotic cell cycle (GO:0051321) 3/16 2.70E-04 1.27E-02 -2.43 10.62 DMC1;HORMAD2;HORMAD1oogenesis (GO:0048477) 2/9 2.26E-03 8.82E-02 -2.41 5.86 CCDC36;HORMAD1cytoskeleton organization (GO:0007010) 4/91 6.42E-03 1.70E-01 -2.62 4.65 FMNL3;STRIP2;FGD6;SIPA1L3meiotic DNA double-strand break formation (GO:0042138) 3/5 5.16E-06 3.02E-04 -0.52 4.18 SPO11;CCDC36;HORMAD1synapsis (GO:0007129) 2/10 2.81E-03 9.40E-02 -1.58 3.74 SYCP1;CCDC36female gamete generation (GO:0007292) 2/16 7.26E-03 1.70E-01 -2.11 3.74 SPO11;DMC1regulation of exocytosis (GO:0017157) 2/16 7.26E-03 1.70E-01 -2.06 3.64 RIMS2;STXBP5L

D Ythdc2 -/- downregulated genes: enriched GO terms in the Biological Process ontology

gene

exon

sens

e

gene

exon

antis

ense

gene

intro

n sen

se

gene

intro

n anti

sens

e

repe

at se

nse

repe

at an

tisen

se

none

miRN

A se

nse

miRN

A an

tisen

se

0

10

20

30

40%

of re

ads

D

transcript parts: 5' −> 3'

norm

alize

d rea

d cov

erag

e

0 20 40 60 80 100

0

20

40

60

80

100

transcript parts: 5' −> 3' 0 20 40 60 80 100

0

20

40

60

80

100

transcript parts: 5' −> 3' 0 20 40 60 80 100

0

20

40

60

80

100

Ythdc2+/+P8 P12 P20

Ythdc2+/+Ythdc2+/-

inputm6A IP

inputm6A IP

inputm6A IP

norm

aliz

ed re

ad c

over

age

0 20 40 60 80 1000

20406080

100120140

0 20 40 60 80 100 0 20 40 60 80 100

0 20 40 60 80 100

020406080

100120140

0 20 40 60 80 100 0 20 40 60 80 100

exon parts: 5' −> 3' exon parts: 5' −> 3' exon parts: 5' −> 3'

inputm6A IP

inputm6A IP

P12

Ythd

c2+/

-P2

0Yt

hdc2

+/+

B

Figure-S5

gene

exon

sens

e

gene

exon

antis

ense

gene

intro

n sen

se

gene

intro

n anti

sens

e

repe

at se

nse

repe

at an

tisen

se

none

miRN

A se

nse

miRN

A an

tisen

se

0

10

20

30

40

Ythdc2 -/-Ythdc2 +/-Ythdc2 +/+

% of

read

s

C

E

+/- vs. +/+

Gene expression-/- vs. +/+ -/- vs. +/-

●

● YTHDC2padj < 0.1

F

0 5 10 15

−1.0

−0.5

0.0

0.5

1.0


Fold

cha

nge

(log2

)

●●

●

●

●

0 5 10 15

−1.0

−0.5

0.0

0.5

1.0


Fold

cha

nge

(log2

)

●

●

●

●

●

● ●

●

●

0 5 10 15

−1.0

−0.5

0.0

0.5

1.0


Fold

cha

nge

(log2

)

●

●

●

●

●

●

●

●

●●●

●

●

AladRpl21

AladRpl21

Gene expression+/

+ +/- -/- -/-

AladRpl21Etnk2Sft2d2Ren1AcadlRnaselWdfy1Akap3Tnp1Gsg1Spz1Akap4Prm2Prm1Odf1Tnp2Smcp

−1−0.5 0 0.5 1Row Z−Score

Color Key

P8 testesSS

C_1

SSC_

2

SC_1

SC_2

RS_1

RS_2

4250 m6A enriched genes−2 0 2

Color Key

Row Z−Score

G

first exon before last exon last exon

A

0

1

2

3

4

bits

1

GTA

2

AG

3

GA

4

C5

AT

6

A

T

G7

CGAT

8

CTAG

21 nt sequences centered on m6A IP peak summitsMEME motif search

Supplemental Table 1. List of all deep-sequencing libraries created in this study. Related to STAR Methods and Figure 3 and 4

sample description reads

RR439 RNA Bind-N-Seq: Bead control for YTH IP from YTHDC2-Sample1 29739568




RR443 RNA Bind-N-Seq: YTH IP from YTHDC2-sample1 23125095




sample description mm10 mapped reads

RR514 P12 testicular total RNA sequencing: Ythdc2 -/- 47780337

RR515 P12 testicular total RNA sequencing: Ythdc2 +/- 55642601









RR523 P8 testicular total RNA sequencing: Ythdc2 +/+ 58413597


RR524 P8 testicular m6a IP: input: Ythdc2 +/+ 33333536

RR525 P12 testicular m6a IP: input: Ythdc2 +/- 33553370

RR526 P12 testicular m6a IP: input: Ythdc2 +/- 29995122



RR529 P8 testicular m6a IP: IP: Ythdc2 +/+ 35327563

RR530 P12 testicular m6a IP: IP: Ythdc2 +/- 34087212

RR531 P12 testicular m6a IP: IP: Ythdc2 +/- 39296380




RR534 P0 ovarian total RNA sequencing: Ythdc2 -/- 27163873

RR535 P0 ovarian total RNA sequencing: Ythdc2 +/- 27321804

RR536 P0 ovarian total RNA sequencing: Ythdc2 +/- 33712411

RR537 P0 ovarian total RNA sequencing: Ythdc2 -/- 27783885

Supplemental Table S3. Genes with significantly different expression in P8 testes of Ythdc2-/-

vs Ythdc2+/-

and Ythdc2-/-

vs Ythdc2+/+

.

Related to STAR Methods and Figure 4.

name HET vs. WT KO vs. WT KO vs. HET HET vs. WT KO vs. WT KO vs. HET

Prm2 0.74 -0.29 -1.03 1.47E-04 1.00E+00 4.30E-09

Akap4 0.52 -0.21 -0.73 8.53E-02 1.00E+00 1.29E-04

Prm1 0.51 -0.19 -0.70 2.26E-02 1.00E+00 5.00E-05

Tnp2 0.45 -0.22 -0.67 8.53E-02 1.00E+00 6.52E-05

Gsg1 0.43 -0.12 -0.55 1.45E-01 1.00E+00 9.22E-03

Akap3 0.45 -0.08 -0.52 1.02E-01 1.00E+00 3.11E-02

Wdfy1 -0.28 -0.77 -0.49 1.00E+00 1.36E-02 1.00E+00

Tnp1 0.35 -0.12 -0.47 9.00E-01 1.00E+00 5.67E-02

Spz1 0.34 -0.11 -0.45 5.55E-01 1.00E+00 3.40E-02

Odf1 0.28 -0.14 -0.42 1.00E+00 1.00E+00 4.69E-02

Smcp 0.28 -0.11 -0.39 1.00E+00 1.00E+00 5.89E-02

Acadl 0.36 0.81 0.45 1.00E+00 5.31E-03 1.00E+00

Etnk2 0.41 0.87 0.45 1.00E+00 5.27E-04 1.00E+00

Ren1 0.52 0.99 0.46 1.00E+00 3.63E-05 1.00E+00

Sft2d2 0.40 0.90 0.49 1.00E+00 3.36E-04 1.00E+00

Rnasel 0.59 1.15 0.56 8.60E-01 2.05E-07 7.09E-01

Alad 0.02 0.70 0.68 1.00E+00 5.07E-02 5.67E-02

Rpl21 -0.06 0.89 0.95 1.00E+00 2.68E-04 4.97E-05

log2 fold change adjusted p-value

Table S5: List of DNA primers and RNA oligonucleotides used in this study.

Name Sequence Used for

DNA primers

RRoligo807 GCCTGTCTCATGGGAAGCAT Genotyping PCR for Ythdc2

RRoligo808 ATCCAGCAGTGTTTCCTGTT Genotyping PCR for Ythdc2

RRoligo792

5’-

GAAATTAATACGACTCACTATAGGATAGTCGTACTAT

ATATCACGTTTTAGAGCTAGAAATAGC-3’

CRISPR F primer (gene-

specific sequence in red)

RRoligo793

5’-

GAAATTAATACGACTCACTATAGGTTCATGCACTTCA

TCCTGCAGTTTTAGAGCTAGAAATAGC-3’

CRISPR F primer (gene-

specific sequence in red)

CRISPR sgR primer

3’CAAAATCTCGATCTTTATCGTTCAATTTTATTCCG

ATCAGGCAATAGTTGAACTTTTTCACCGTGGCTCAGC

CACGAAAA-5’

Common reverse primer for

preparing template for

guide RNA

RNA Oligos

RPRNA18 AGCACCGUAAAGACGC 3' overhang duplex

RPRNA19

GCGUCUUUACGGUGCUUAAAACAAAACAAAACAAAAC

AAA 3' overhang duplex

RPRNA20 GCGUCUUUACGGUGCU 5' overhang duplex

RPRNA21

AACAAAACAAAACAAAACAAAAUAGCACCGUAAAGAC

GC 5' overhang duplex

RPRNA28 GGGAGCGUUCCGUCUGCUAUUAUCACG EMSA

RPRNA29 GAUAAGCUGUAGGAGGUUCUUCUAGUU EMSA

RPRNA31 GCGCGAUCGAUCGUUGCUGACUGAGCC EMSA

6-FAM RNA GAACCGGXCUGUCUUA X= A, for anisotropy expt

6-FAM m6A RNA GAACCGGXCUGUCUUA

X= m6A, for anisotropy

expt

MMRNA4

NNNNNNNNNNXNNNNNNNNNNAGATCGGAAGAGCACA

CGTCT

X= m6A, N=randomized, for

ATPase assay

Magdalena Natalia Wojtas, Radha Raman Pandey, Mateusz ... · Sachidanandam, and Ramesh S. Pillai....

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