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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
F
A B
0
100
200
300
400
500
600
0 5 10 15 20 25
S75 10/300 GLSUMO-hYTHDC2 YTH domain
mAU
ml
A
position
frequ
ency
(%)
1 3 5 7 9 11 13 15 17 19 21
1015
2025
3035
40
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position1 3 5 7 9 11 13 15 17 19 21
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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
70
100
120
55
40
35
hYTHDC2 ∆YTH
Instant Blue stain
kDa
180
70
100
120
55
40
S200 gel-filtration elutions
1015
2025
3035
40
1015
2025
3035
40
1015
2025
3035
40
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
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
% 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
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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
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e
repe
at se
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repe
at an
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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
Mean of normalized counts (log2)
Fold
cha
nge
(log2
)
●●
●
●
●
0 5 10 15
−1.0
−0.5
0.0
0.5
1.0
Mean of normalized counts (log2)
Fold
cha
nge
(log2
)
●
●
●
●
●
● ●
●
●
0 5 10 15
−1.0
−0.5
0.0
0.5
1.0
Mean of normalized counts (log2)
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
RR440 RNA Bind-N-Seq: Bead control for YTH IP from YTHDC2-Sample2 25680614
RR441 RNA Bind-N-Seq: Bead control for YTH IP from YTHDC2-Sample3 25227457
RR442 RNA Bind-N-Seq: Bead control for YTH IP from YTHDC2-Sample4 23092098
RR443 RNA Bind-N-Seq: YTH IP from YTHDC2-sample1 23125095
RR444 RNA Bind-N-Seq: YTH IP from YTHDC2-sample2 23617600
RR445 RNA Bind-N-Seq: YTH IP from YTHDC2-sample3 20809839
RR446 RNA Bind-N-Seq: YTH IP from YTHDC2-sample4 21959602
sample description mm10 mapped reads
RR514 P12 testicular total RNA sequencing: Ythdc2 -/- 47780337
RR515 P12 testicular total RNA sequencing: Ythdc2 +/- 55642601
RR516 P12 testicular total RNA sequencing: Ythdc2 +/- 44672418
RR517 P12 testicular total RNA sequencing: Ythdc2 +/- 41866756
RR518 P12 testicular total RNA sequencing: Ythdc2 -/- 46374945
RR519 P12 testicular total RNA sequencing: Ythdc2 -/- 48594481
sample description mm10 mapped reads
RR520 P8 testicular total RNA sequencing: Ythdc2 -/- 48259151
RR521 P8 testicular total RNA sequencing: Ythdc2 -/- 53354894
RR522 P8 testicular total RNA sequencing: Ythdc2 +/- 47781419
RR523 P8 testicular total RNA sequencing: Ythdc2 +/+ 58413597
sample description mm10 mapped reads
RR524 P8 testicular m6a IP: input: Ythdc2 +/+ 33333536
RR525 P12 testicular m6a IP: input: Ythdc2 +/- 33553370
RR526 P12 testicular m6a IP: input: Ythdc2 +/- 29995122
RR527 P20 testicular m6a IP: input: Ythdc2 +/+ 35691697
RR528 P20 testicular m6a IP: input: Ythdc2 +/+ 33880816
RR529 P8 testicular m6a IP: IP: Ythdc2 +/+ 35327563
RR530 P12 testicular m6a IP: IP: Ythdc2 +/- 34087212
RR531 P12 testicular m6a IP: IP: Ythdc2 +/- 39296380
RR532 P20 testicular m6a IP: IP: Ythdc2 +/+ 38050174
RR533 P20 testicular m6a IP: IP: Ythdc2 +/+ 35328608
sample description mm10 mapped reads
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