Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development

The N6-methyladenosine (m6A) modification is the most prevalent internal RNA modification in eukaryotes. The majority of m6A sites are found in the last exon and 3’ UTRs. Here we show that the nuclear m6A reader YTHDC1 is essential for embryo viability and germline development in mouse. Specifically, YTHDC1 is required for spermatogonial development in males and for oocyte growth and maturation in females; Ythdc1-deficient oocytes are blocked at the primary follicle stage. Strikingly, loss of YTHDC1 leads to extensive alternative polyadenylation in oocytes, altering 3’ UTR length. Furthermore, YTHDC1 deficiency causes massive alternative splicing defects in oocytes. The majority of splicing defects in mutant oocytes are rescued by introducing wild-type, but not m6A-binding-deficient, YTHDC1. YTHDC1 is associated with the pre-mRNA 3’ end processing factors CPSF6, SRSF3, and SRSF7. Thus, YTHDC1 plays a critical role in processing of pre-mRNA transcripts in the oocyte nucleus and may have similar non-redundant roles throughout fetal development.

We previously identified YTHDC1 as a meiotic chromatin-associated protein in a proteomic screen [57]. YTHDC1 (initially referred to as YT521-B) changes alternative splicing patterns in a concentration-dependent manner [27] and localizes to nuclear speckles, which contain active transcription sites [28]. Tyrosine phosphorylation of YTHDC1 regulates its intra-nuclear localization, thereby modulating its effects on alternative splicing [58]. YTHDC1 promotes exon inclusion by recruitment of serine/arginine-rich (SR) splicing factor 3 (SRSF3), a pre-mRNA splicing factor [46]. YTHDC1 facilitates nuclear export of m 6 A-containing mRNAs through SRSF3 and NXF1 [59]. Although these studies in cultured cells have provided important insights into the function of YTHDC1, its requirement during development is unknown. In addition, the biological function of accumulation of m 6 A sites in 3' UTRs remains mysterious. Here, we report that YTHDC1 is essential for embryonic development in the mouse. Using a conditional inactivation approach, we find that YTHDC1 is required for survival of spermatogonia in males and controls postnatal oocyte development in females. Strikingly, in addition to alternative splicing defects, loss of YTHDC1 causes widespread alternative polyadenylation in oocytes. Importantly, YTHDC1 is associated with SR proteins and pre-mRNA 3' end processing factors.

Nuclear localization of YTHDC1 in male germ cells, oocytes and preimplantation embryos
We examined expression of YTHDC1 in adult mouse tissues using polyclonal antibodies raised against an N-terminal region of mouse YTHDC1 (S2A Fig). Western blot analysis showed that YTHDC1 was expressed in multiple adult mouse tissues including brain, testis, and ovary, with an apparent molecular weight of~120 kDa (Fig 1A). High levels of YTHDC1 were present in postnatal oocytes, MII eggs, and pre-implantation embryos, and low levels in germinal vesicle (GV) stage oocytes ( Fig 1B). The increase in YTHDC1 protein abundance between the GV oocyte stage and MII egg suggests that YTHDC1 is encoded by a dormant maternal mRNA that is recruited during oocyte maturation. Immunostaining showed that YTHDC1 localized to the nucleus in postnatal oocytes and pre-implantation embryos, with the increase in staining between the GV oocyte and MII egg being consistent with the immunoblotting results (Fig 1C). The diffuse cytoplasmic signal of YTHDC1 and its increased abundance in MII oocytes suggest that Ythdc1 is under translational control, possibly in preparation for zygotic activation at the two-cell stage. The nuclear localization of YTHDC1 is consistent with a previous finding that it is associated with chromatin [57]. Notably, in postnatal day (PND) 5 and 12 oocytes, transcription is active and YTHDC1 is nuclear. In adult testis (S1 Fig), YTHDC1 is nuclear in spermatogonia, spermatocytes, and round spermatids, which are transcriptionally active. However, YTHDC1 is absent in elongating and elongated spermatids, which are transcriptionally inactive due to nuclear condensation (S1 Fig). Therefore, the nuclear localization of YTHDC1 in cells with active transcription suggests that it is involved in co-transcriptional and/or post-transcriptional regulations.

YTHDC1 is required for embryo viability
To determine the physiological function of Ythdc1, we generated a Ythdc1 floxed (conditional) allele, referred to as Ythdc1 fl , by gene targeting in embryonic stem (ES) cells (S2B Fig). Ythdc1 fl/fl mice were healthy and fully fertile. We next crossed Ythdc1 fl/fl mice to Actb-Cre mice, which express Cre ubiquitously, to obtain mice with a Ythdc1 null allele (Ythdc1 +/-) [60]. Cre-mediated excision of the floxed exons removes the YTH domain and causes a frameshift in the resulting Ythdc1 mutant transcript (S2B Fig). Intercrosses of Ythdc1 +/mice did not produce any Ythdc1 -/pups, suggesting that Ythdc1 is essential for embryonic development (S2C Fig). To determine the time of developmental failure, we genotyped fetuses recovered from intercrosses of Ythdc1 +/mice at embryonic day 8.5 (E8.5), E9.5, and E11.5. No Ythdc1 -/embryos were found at E11.5. Out of 9 embryos at E8.5 and out of 33 embryos at E9.5, only one Ythdc1 -/embryo was found at each time point (S2C Fig). Resorbed embryos were found at E8.5 through E11.5 and expected to be homozygous mutants based on their Mendelian distribution (S2C Fig). These results show that YTHDC1 is indispensable for embryo development past early post-implantation stages.

YTHDC1 is essential for spermatogonium survival and male fertility
To bypass the embryonic lethality resulting from Ythdc1 deficiency, we used Ddx4-Cre to inactivate Ythdc1 specifically in the germline to generate Ythdc1 fl/-Ddx4-Cre (referred to as Ythdc1 cKO ) mice (Figs 2 and 3A). All subsequent studies were conducted with Ythdc1 fl/-Ddx4-Cre (cKO) mice unless noted otherwise. Ddx4-Cre expression begins at~E15 in both male and female germ cells but differs in the developmental stage of onset due to the sexual dimorphism in the timing of meiotic entry [61]. In males, Ddx4-Cre is expressed in mitotic germ cells including spermatogonia prior to meiosis, whereas in oocytes, Ddx4-Cre expression occurs only after meiotic entry (Fig 3A). Ythdc1 cKO mice were viable and grossly normal. Essential role of YTHDC1 in female fertility Seminiferous tubules from newborn (PND0) Ythdc1 cKO males contained prospermatogonia (Fig 2A), which lacked YTHDC1 as determined by immunostaining (S3 Fig). Tubules from PND8 Ythdc1 cKO males contained substantially fewer spermatogonia than those from control Ythdc1 fl/+ or Ythdc1 fl/males ( Fig 2B). However, testes from PND25 and adult Ythdc1 cKO Essential role of YTHDC1 in female fertility males lacked any germ cells including mitotic spermatogonia and exhibited a Sertoli-cell-only phenotype (Fig 2C and 2D), demonstrating that Ythdc1 is required for development of spermatogonia and male fertility.

Inactivation of YTHDC1 causes oocyte maturation arrest and female sterility
In contrast to the absence of germ cells in adult Ythdc1 cKO testis, oocytes were present in ovaries from 8-week-old Ythdc1 fl/-Ddx4-Cre (cKO) females (Fig 3). Wild-type adult ovaries contained follicles at different developmental stages, including primary, secondary, and antral follicles (Fig 3B and 3C). However, Ythdc1 fl/-Ddx4-Cre ovaries lacked secondary or antral follicles, indicating that oocyte development was blocked at the primary follicle stage, which is characterized by one layer of granulosa cells surrounding the oocyte (Fig 3B). Histological analysis of ovaries from older Ythdc1 fl/-Ddx4-Cre females (6-month and beyond) showed a complete loss of oocytes. Western blot analysis confirmed that YTHDC1 protein was absent in oocytes collected from Ythdc1 fl/-Ddx4-Cre ovaries (S4A Fig). As expected, a nuclear immunofluorescent signal of YTHDC1 was not detected in Ythdc1 mutant oocytes (S4B Fig). These results confirm the specificity of our YTHDC1 antibody and the complete depletion of YTHDC1 in Ythdc1 fl/-Ddx4-Cre oocytes.
Because expression of Ddx4-Cre begins at the pachytene stage of meiotic prophase I during fetal development, it is not clear whether the observed defects in Ythdc1 fl/-Ddx4-Cre postnatal ovaries were due to the requirement of YTHDC1 at embryonic or postnatal stages. To investigate whether postnatally expressed YTHDC1 is required for oocyte development, we used Zp3-Cre to inactivate Ythdc1 in oocytes postnatally ( Fig 3A). Zp3-Cre is expressed in developing oocytes around postnatal day 3 ( Fig 3A) [62]. We found that Ythdc1 fl/-Zp3-Cre ovaries exhibited similar defects in folliculogenesis as observed in Ythdc1 fl/-Ddx4-Cre ovaries-blockade at the primary follicle stage (Fig 3C). We next performed mating tests of three Ythdc1 fl/-Zp3-Cre females and three wild-type littermate control females. At the age of 8 weeks, each female was housed with one wild-type male for two months. The three control females produced two litters each (6.8 ± 1.4 pups/litter), whereas none of the three Ythdc1 fl/-Zp3-Cre females produced any offspring. Taken together, these genetic studies demonstrate that YTHDC1 plays an essential role in postnatal oocyte development.

Ythdc1-deficient oocytes contain large cytoplasmic RNA granules
We were able to retrieve oocytes from ovaries of Ythdc1 fl/-Ddx4-Cre females at the ages of 3-6 weeks by poking. However, the number of oocytes retrieved from Ythdc1 fl/-Ddx4-Cre females was only 10% (n = 3) that of wild-type littermates. In addition, the GV oocytes from Ythdc1 fl/-Ddx4-Cre females were not able to resume meiosis in vitro. In contrast to the smooth appearance of wild-type germinal vesicle (GV) stage oocytes, Ythdc1-deficient oocytes contained one or two prominent granules in the cytoplasm (Fig 4A). Such granules were not observed in wild-type oocytes. These granules stained positive with Sytox green, which recognizes both DNA and RNA, suggesting a nucleic acid content (Fig 4B). When double stained with both DAPI and Sytox green, the nuclei of Ythdc1-deficient oocytes were positive for both stains, whereas the granules only retained the Sytox green stain, indicating that the granules contained RNA but not DNA (Fig 4B). To our knowledge, such large RNA granules have not been observed before. The appearance of large cytoplasmic RNA granules indicates severe defects in RNA metabolism in oocytes in the absence of YTHDC1. It is possible that, like P granules, incorrectly processed RNAs are sequestered in these novel RNA granules in oocytes in the absence of YTHDC1. Knockdown of YTHDC1 in HeLa cells causes acute nuclear accumulation of mRNAs within hours [59]. We did not observe nuclear accumulation of RNAs in postnatal Ythdc1-deficient oocytes, possibly because inactivation of Ythdc1 begins at E15 (Fig 3A), weeks prior to our analysis.
To investigate the consequences of Ythdc1 deficiency on the oocyte transcriptome, we performed RNA-seq analysis of oocytes collected from 6-week-old wild-type and Ythdc1 fl/-Ddx4-Cre females (S1 Table). With a FDR cutoff of 0.01, a total of 4933 transcripts showed differential expression: 2656 transcripts were up-regulated and 2277 transcripts down-regulated in Ythdc1-deficient oocytes compared with control oocytes, indicating that the transcriptome in Ythdc1-deficeint oocytes was dramatically altered (S5A Fig and S2 Table).

Widespread splicing defects in Ythdc1-deficient oocytes
Because YTHDC1 affects alternative splicing in cultured somatic cells [27], we next analyzed the oocyte RNA-seq data to systematically identify local splicing variants (LSVs) between wildtype and Ythdc1-deficient oocytes using the MAJIQ package [63]. We identified a total of 2937 significant LSVs (q < 0.05) with ΔPSI (difference in percent spliced in) > 0.2 ( Fig 5A and S3  Table). These LSVs affected 1966 genes, involved 10,266 exons, and included differential retention of 500 introns. Of the 1966 genes with LSVs, 34% (659 genes) were differentially expressed between wild-type and Ythdc1-deficient oocytes (up-regulated, 245 genes; down-regulated, 414 genes). According to GO analysis, these changes affect genes involved in multiple fundamental biological processes, including chromatin modification, regulation of transcription, mRNA processing, and regulation of translation (S6 Fig).
We designed RT-PCR assays to validate different types of MAJIQ-identified splicing events: exon inclusion/skipping, intron retention, and splicing in 3' UTRs. Using GV-stage oocytes from wild-type and Ythdc1 fl/-Ddx4-Cre mice, RT-PCR analysis confirmed 90% (9 of 10 tested) of LSVs involving internal exons (Fig 5B and 5C). For example, as illustrated in the gene track view, the second exon (part of the 5'UTR) of Tmem2 was partially skipped in wild-type but not in Ythdc1-deficient oocytes ( Fig 5B). Two LSVs affecting Jam2 and Spata7, respectively, involved nearly complete exon skipping in wild-type oocytes but partial exon skipping in Ythdc1-deficient oocytes. Exon skipping in two genes (Dner and Rad1) was complete in Ythdc1-deficient oocytes but partial in wild-type. Four LSVs in Enpp5, Hip1r, Rap1a, and Parp6 resulted in partial exon skipping in Ythdc1-deficient oocytes whereas no skipping occurred in wild-type. There was no apparent preference for the directionality of exon skipping in regard to genotype. In total, our validation results suggest that most LSVs predicted by MAJIQ are true splicing events.
LSVs involving introns or 3' UTRs were more complex. We tested nine intron-retention LSVs predicted by MAJIQ and confirmed six of these (67%) by RT-PCR (Fig 6A and 6B). Among three confirmed LSVs, two transcripts (Dnpep and Mcph1) showed intron retention preferentially in wild-type oocytes, whereas one mRNA (Phf1) retained introns preferentially in Ythdc1-deficient oocytes. We examined 13 MAJIQ-predicted LSVs involving splicing within 3' UTRs and validated three of these (23%) by RT-PCR (Fig 6C and 6D): Ifnar1, Abl2, and Ikzf5. Coincidentally, all of these transcripts were associated with longer 3' UTRs in wild-type oocytes. Although MAJIQ was not designed for the analysis of changes in introns and 3' UTR length as pointed out by the MAJIQ authors, we were able to validate most of the MAJIQ-predicted LSVs involving introns and some of the 3' UTR splicing events.
We further examined the 9 LSVs involving exon inclusion/skipping using GV-stage oocytes from 6-week-old Ythdc1 fl/-Zp3-Cre and wild-type females. All 9 LSVs validated in Ythdc1 fl/-Ddx4-Cre oocytes ( Fig 5) were also confirmed in Ythdc1 fl/-Zp3-Cre oocytes (S7 Fig). Collectively, our results show that inactivation of YTHDC1 in oocytes causes severe defects in mRNA splicing. Essential role of YTHDC1 in female fertility

Extensive alternative polyadenylation in Ythdc1-deficient oocytes
The majority of m 6 A sites are present in the 3' most exons, raising the possibility that m 6 A may play a role in regulating 3' UTR length [5,9]. Many genes produce transcripts with 3' UTRs of different lengths due to usage of alternative polyadenylation (APA) sites and 3' UTRs contain sites for microRNAs and RNA-binding proteins. Thus, the 3' UTR of a particular mRNA regulates its translation and subcellular localization [64,65]. For instance, transcripts in brain exhibit extensive lengthening of 3' UTRs due to APA [66]. We systematically analyzed the 3' UTR length of wild-type versus Ythdc1-deficient oocytes using the ROAR algorithm [67]. The ROAR program identifies alternative polyadenylation using standard RNA-seq data by measuring the reads upstream (pre) and downstream (post) of the annotated polyadenylation site (PAS) (Figs 7A and 6B). ROAR analysis of our oocyte RNA-seq data revealed 1210 alternative polyadenylation (APA) events in 864 genes between wild-type and Ythdc1 mutant oocytes (cutoff, p value < 0.05; Fig 7A and S4 Table). Some genes had more than one differential APA event. Overall, 709 APA events (ROAR < 1) resulted in higher levels of the longer isoform (longer 3' UTR) in Ythdc1-deficient oocytes, whereas 501 APA events (ROAR > 1) was associated with higher levels of the shorter isoform in the mutant (Fig 7A).
We chose 8 transcripts with predicted longer 3' UTRs in the Ythdc1 mutant for RT-PCR validation ( Fig 7B). These 8 transcripts were not differentially expressed between wild-type and mutant oocytes. Our validation strategy involved one PCR assay (termed PRE) that amplified both short and long transcripts and a second PCR assay (termed POST) that was specific for the long isoform. The POST RT-PCR assay for Arl5a produced a stronger signal from Ythdc1-deficient versus wild-type oocytes, indicating that the former contained a higher level of the long isoform. Of the eight transcripts tested, seven (88%) (Arl5a, Ddx21, Noc3l, Rybp, Scamp1, Slc11a2, and Slc25a51) preferentially produced the longer isoform in Ythdc1-deficient oocytes due to APA, whereas, one transcript (Frs2) did not exhibit detectable differences in isoform prevalence using this assay (Figs 7C and 6D). These results demonstrate extensive alternative polyadenylation in Ythdc1-deficient oocytes. Previous findings in brain tissue [9] have shown that five of seven gene transcripts with APA defects contained known m 6 A sites in the last exons: Arl5a, Ddx21, Noc3l, Slc11a2, and Slc25a51, implicating m 6 A in regulation of alternative polyadenylation.

m 6 A-dependent rescue of alternative splicing defects in Ythdc1-deficient oocytes
To examine the effect of m 6 A on splicing in oocytes, we collected oocytes from wild-type and Ythdc1 fl/-Ddx4-Cre ovaries at postnatal day 12 (PND12), when oocytes are still transcriptionally active. We evaluated alternative splicing of the nine transcripts for which we had identified splicing defects in GV stage oocytes from 6-week-old Ythdc1 fl/-Ddx4-Cre mice ( Fig 5) and found that all nine transcripts showed similar splicing defects in PND12 Ythdc1-deficient oocytes (Fig 8, first two lanes of center panel). However, there were notable differences for two transcripts: Rad1 and Tmem2. The Rad1 two-exon-skipping isoform was detected in PND12 mutant oocytes (Fig 8) but not in 6-week-old mutant oocytes (Fig 5). Similarly, the Tmem2 spliced short isoform was present in PND12 mutant oocytes but absent in 6-week-old oocytes. These data suggest that the short isoforms of Rad1 and Tmem2 were degraded during the long period between cessation of transcription at PND20 and the time point of analysis at 6 weeksof-age.
To investigate if alternative splicing defects in Ythdc1-deficient oocytes could be rescued by supplying YTHDC1, we used transcriptionally active PND12 oocytes. We injected PND12 Ythdc1-deficient oocytes with in vitro transcribed wild-type or mutant Ythdc1 mRNA, followed by overnight culture. The Ythdc1 mutant mRNA contains two missense mutations (W377A, W428A) that completely abolish the m 6 A binding activity of YTHDC1 [24]. We quantified the FLAG-YTHDC1 protein levels in the nucleus of injected Ythdc1-deficient Essential role of YTHDC1 in female fertility oocytes by immunofluorescence and confocal microscopy and found no difference in Essential role of YTHDC1 in female fertility YTHDC1 protein levels between oocytes (4 oocytes each) injected with wild-type and mutant Ythdc1 mRNAs. We found that exon skipping and exon inclusion defects in six transcripts were rescued in Ythdc1-deficient oocytes injected with wild-type but not mutant Ythdc1 mRNA, whereas no difference was observed for the remaining three genes (Dner, Enpp5 and Jam2) (Fig 8). These rescue experiments suggest that the majority of alternative splicing defects in Ythdc1-deficient oocytes is m 6 A-dependent. However, we cannot rule out the possibility that the failure of mutant YTHDC1 ((W377A, W428A) to rescue might be caused by reduced RNA-binding activity or instability, independent of m 6 A.

YTHDC1 interacts with pre-mRNA 3'end processing factors
To elucidate the mechanism by which YTHDC1 affects alternative polyadenylation, we investigated potential interactions of YTHDC1 with pre-mRNA 3'end cleavage and polyadenylation factors by co-immunoprecipitation (Fig 9). Cleavage factor Im (CFIm) and cleavage stimulating factor (CSTF) are two multi-protein complexes that bind to upstream sequence elements (USE) and downstream sequence elements (DSE) around the PAS, respectively [68,69]. We found that YTHDC1 was associated with CPSF6, one of the four subunits of the CFIm complex ( Fig 9A). This result is consistent with previous reports identifying CPSF6 among proteins co-immunoprecipitated with human YTHDC1 in 293T cells [46]. However, YTHDC1 did not interact with NUDT21, another subunit of the CFIm complex ( Fig 9A). In addition, YTHDC1 was not associated with cleavage stimulating factors CSTF1 or CSTF2 by co-transfection and co-IP assays. Interestingly, knockdown of Cpsf6 induces widespread use of proximal PAS, resulting in 3' UTR shortening [70,71]. Moreover, a mutation in the Medaka Cpsf6 gene causes 3' UTR shortening in developing embryos and a defect in primordial germ cell migration [72].
YTHDC1 interacts with the SR splicing factors SRSF3 and SRSF7 (Fig 9B). SRSF3 and SRSF7 couple RNA processing with mRNA export through association with the nuclear mRNA export factor NXF1 [73]. SRSF3 and SRF7 bind to the last exons and regulate polyadenylation in an opposing manner. Knockdown of SRSF3 leads to 3' UTR shortening, whereas depletion of SRSF7 results in 3' UTR lengthening [73]. In conclusion, these results support a model in which YTHDC1 regulates alternative polyadenylation through interaction with the 3'end processing machinery.

Discussion
Here, we report that the nuclear m 6 A reader YTHDC1 is essential for mouse embryogenesis and germline development, and describe a critical role of YTHDC1 in orchestrating m 6 Adependent processing of pre-mRNA transcripts in oocytes. Our studies implicate YTHDC1 in the choice of polyadenylation sites, which determines the length of 3' UTRs. The 3' UTR contains target sites for microRNAs and many RNA-binding proteins. Therefore, lengthening or shortening of 3' UTR would predictably have profound effects on translation efficiency, transcript stability, and subcellular transcript localization [64,65,68,69]. Precise translational control of maternal transcripts is especially critical during oocyte maturation, due to lack of transcription during this prolonged stage. We find that loss of YTHDC1 in oocytes results in alternative polyadenylation and thus altered 3' UTR length in more than 800 genes. To date, YTHDC1 is the only m 6 A reader that has been demonstrated to regulate 3' UTR length.
Triple knockdown of three m 6 A writer components (METTL3, METTL14, and WTAP) in human A549 cells changes the usage of proximal versus distal polyadenylation sites with some switching to proximal sites and others switching to distal sites, demonstrating a critical role for m 6 A in regulation of 3' UTR length [9]. In addition, ALKBH5, an m 6 A demethylase, regulates 3'UTR length in male germ cells [74]. How the m 6 A signal is relayed to the 3' end processing machinery is unknown. A number of multi-protein complexes participate in pre-mRNA 3' end cleavage and polyadenylation, including cleavage factor Im (CFIm), cleavage and polyadenylation specificity factor (CPSF), cleavage stimulating factor (CSTF), and poly(A)-binding proteins [68,69]. We find that YTHDC1 forms complexes with components of the 3' end processing machinery: CPSF6 (a CFIm component), SRSF3, and SRSF7 (Fig 9). These factors bind to the 3' UTR around the PAS. Specifically, the CFIm binds to the UGUA motif upstream of the PAS. Knockdown of each of these factors in cell culture causes a shift in PAS usage, resulting in APA. Knockdown of CPSF6 favors usage of proximal PAS [70,71], whereas knockdown of SRSF7 causes preferential usage of distal PAS [73]. Our data support a model in which YTHDC1 recognizes m 6 A in the last exons of pre-mRNA transcripts and orchestrates the choice of polyadenylation sites through interactions with 3'end processing factors. YTHDC1 may recruit these factors to the 3' UTRs or sequester them in the nucleoplasm through interactions, resulting in opposing APA patterns. Alternatively, these factors may compete for binding to YTHDC1. In addition, SRSF3 and SRSF7 link alternative polyadenylation with nuclear export through interaction with NXF1 [73]. Therefore, it is conceivable that, through interaction with SRSF3 and SRSF7, YTHDC1 may couple m 6 A in alternatively polyadenylated transcripts with nuclear export. In cultured cells, YTHDC1 facilitates binding of m 6 A-modified nuclear transcripts to SRSF3 and NXF1 and mediates nuclear export [59].
Our study reveals an essential role for YTHDC1 in development of both the embryo and the germline. Proteins (writers, readers, and erasers) involved in establishment, recognition, and erasure of m 6 A sites in mRNAs play important roles in development and fertility in mouse. Loss of the key m 6 A writer enzyme METTL3 causes early post-implantation lethality with defects in lineage priming [49]. m 6 A mainly reduces mRNA stability in embryonic stem cells and pre-implantation embryos and its loss leads to a failure in termination of naïve pluripotency during lineage specification [49]. Conditional inactivation of Mettl3/Mettl14 reveals their essential role in spermatogenesis [50,51]. Alkbh5-deficient mice are viable but exhibit Essential role of YTHDC1 in female fertility impaired spermatogenesis with increased apoptosis of meiotic spermatocytes [22]. ALKBH5-mediated m 6 A demethylation affects mRNA export. The cytoplasmic m 6 A reader YTHDC2 interacts with the meiosis-specific protein MEIOC [29,30]. Ythdc2-deficient mice are viable but sterile due to a failure in meiotic progression [26, 31-33]. YTHDC2 together with MEIOC promotes translation efficiency of its target transcripts but decreases their mRNA abundance. In addition, YTHDC2 modulates the level of m 6 A-enriched transcripts in germ cells, which is required for progression through meiosis [32]. Ythdf2 deficiency causes incomplete penetrance of lethality and female-specific infertility [53].
Similar to Mettl3, inactivation of Ythdc1 is embryonic lethal, showing that loss of YTHDC1 is not compensated for by other m 6 A readers. The lack of compensation is not entirely surprising, given that, to date, YTHDC1 is the only known m 6 A reader in the nucleus. By conditional inactivation of Ythdc1 in the germline, we find that YTHDC1 is essential for fertility in both males and females. Specifically, YTHDC1 is required for development of mitotic spermatogonia in males and oocyte growth in females. Because of loss of spermatogonia in Ythdc1 fl/-Ddx4-Cre males, different Cre drivers will be needed to examine its role in meiotic spermatocytes and post-meiotic round spermatids in future studies. Strikingly, the mouse mutant phenotypes of three m 6 A readers YTHDC1, YTHDC2, and YTHDF2 are different, suggesting non-redundant functions. YTHDC2 is required for meiotic progression in both sexes but is dispensable for viability [26, 31-33]. YTHDF2 is partially necessary for viability and specifically required for female fertility and oocyte competence [53]. Here we find that YTHDC1 is essential for viability and is required for spermatogonial development in males and oocyte growth in females. About 200 transcripts were upregulated in Ythdf2-deficient MII oocytes [53]. The overlap between the upregulated transcripts in Ythdf2-deficient oocytes and Ythdc1deficient oocytes is significant (1.48-fold enrichment, p = 0.0004), suggesting that YTHDC1 may play a later role in oocyte competence. In Ythdc1 fl/-Ddx4-Cre or Ythdc1 fl/-Zp3-Cre females, germ cells progress through the prophase of meiosis I, however, oocyte development is blocked at the primary follicle stage. This blockade is similar to the oocyte growth arrest in females lacking GDF9, a key TGFβ receptor ligand [75].
Several early studies using cultured cells show that YTHDC1 is involved in alternative splicing of internal exons in a dosage-dependent manner [27,28,58]. YTHDC1 localizes to socalled YT bodies in the nucleus that contain active transcription sites. Tyrosine phosphorylation of YTHDC1 regulates its solubility in the nucleus and its effect on alternative splicing. Structural demonstration of YTHDC1 as an m 6 A reader raises a possible connection between m 6 A and alternative splicing [24,25]. Among the YTH domain proteins, only YTHDC1 contains a selective binding pocket for the nucleotide preceding the m 6 A nucleotide [25]. Two pre-mRNA splicing factors SRSF3 and SRSF10 competitively bind to YTHDC1 [46]. It was proposed that YTHDC1 promotes exon inclusion by recruiting SRSF3 while blocking SRSF10 binding to target transcripts [46]. In this study, we find that YTHDC1 regulates mRNA splicing in oocytes. In addition, loss of YTHDC1 leads to formation of large novel cytoplasmic RNA-containing granules in the oocyte cytoplasm, which may contain aberrantly processed transcripts. Furthermore, the m 6 A-binding activity of YTHDC1 is required for rescue of alternative splicing defects in Ythdc1-deficient oocytes. Collectively, these in vitro and in vivo studies demonstrate the critical role of YTHDC1 in the regulation of alternative splicing, apparently in an m 6 A-dependent manner.
A number of studies show that m 6 A is a determinant of mRNA stability and turnover in the cytoplasm [23, 47,76]. YTHDC1 facilitates nuclear export of m 6 A-containing mRNAs through its interaction with SRSF3 and thus regulates their cytoplasmic abundance [59]. YTHDC2 regulates the levels of m 6 A-containing transcripts in meiotic germ cells [32]. Binding by YTHDF2 causes redistribution of bound mRNAs to RNA degradation sites [23]. In addition, YTHDF2 regulates maternal mRNA clearance in both zebrafish and mouse [52,53]. In contrast with the established role of m 6 A in mRNA turnover, the role of m 6 A in splicing has been a point of contention in the field. Some studies in ES cells conclude that m 6 A in nascent transcripts has a minor role in splicing, even though Mettl3 inactivation in ES cells affects 3% of~12,000 alternative cassette exons [47,49]. It is possible that Mettl3 inactivation may have more pronounced effect on splicing in differentiated cells. Indeed, Mettl3 inactivation in male germ cells affects splicing [51]. The differentially spliced genes in Ythdc1-deficient oocytes significantly overlap with the differentially spliced genes in Mettl3-deficient testes (S8 Fig). Study of Alkbh5-deficient spermatogenic cells also supports a role of m 6 A in the regulation of splicing [74]. Therefore, the extent of effect on splicing by m 6 A most likely varies in different cell types and developmental stages.

Ethics statement
Mice were maintained and used for experimentation according to the protocol approved by the Institutional Care and Use Committee of the University of Pennsylvania.

Generation of polyclonal antibodies
The GST-YTHDC1 (aa 3-109) fusion protein (S2A Fig) was expressed in E. coli using the pGEX4T-1 vector and affinity purified with glutathione Sepharose 4B (GE Healthcare). Rabbits were immunized with recombinant protein, yielding antisera UP2410 and UP2411 (Cocalico Biologicals Inc.). For western blotting and immunofluorescence, antibodies were affinity purified against the GST fusion protein.
GV oocytes were collected by poking of ovaries or enzymatic digestion. For enzymatic digestion, ovaries were dissected out and placed in Ca 2+ -Mg 2+ -free CZB medium containing 1 mg/ml collagenase (#LS004196, Worthington Biochemical Corp) and 0.2 mg/ml DNase I (Sigma #DN-25) in 35 mm petri dish. Each ovary was chopped into 4-5 pieces. Enzymatic digestion was carried out at 37˚C for 40 min. Ovaries were pipetted up and down several times using a P1000 pipette to facilitate cell dissociation. Oocytes free of follicle cells were transferred and washed with three drops of CZB medium before further analysis.

Western blotting and immunocytochemistry
Equal numbers of GV oocytes, metaphase I (MI) eggs, MII eggs, fertilized eggs and embryos were lysed in 2xSDS loading buffer (Sigma). Lysates were separated by 10% SDS-PAGE gel electrophoresis and proteins transferred to PVDF membrane (Amersham). For western blot analysis of adult mouse tissues, tissue samples were collected from 8-week-old adult mice and 20 μg of protein lysate per tissue analyzed per lane. The following antibodies/antisera were used for western blotting: rabbit anti-YTHDC1 affinity-purified antibody (this study); mouse anti-TUBB antibody (T4026, Sigma), mouse monoclonal ACTB (Clone AC-15, Sigma-Aldrich). Immuno-detection was performed using horseradish peroxidase-conjugated secondary antibodies and ECL prime reagents (Amersham) according to the manufacturer's instructions.
For immunofluorescence, oocyte, egg or embryo samples were fixed in 2.5% paraformaldehyde for 40 min at room temperature. Cells were permeabilized for 15 min in PBS containing 0.2% Triton X-100, blocked in PBS containing 0.2% IgG-free BSA and 0.01% Tween-20 for 30 min (blocking solution), and then incubated with the rabbit anti-YTHDC1 affinity-purified antibody for 1 h at room temperature. After four 15-min washes in blocking buffer, samples were incubated for 1 h with appropriate Cy5-conjugated secondary antibody (Jackson Immu-noResearch). After three additional 15-min washes in blocking buffer, the samples were mounted in Vectashield mounting solution with Sytox green (Vector Laboratories). Images were captured by a Leica TCS SP laser-scanning confocal microscope.
Immunofluorescence analysis in testis was performed as previously described [82]. Briefly, adult or neonatal testes were fixed in 4% formaldehyde for 3-4 h and processed for sectioning in a cryostat. Testicular sections were immunostained with anti-YTHDC1 and anti-SP10 antibodies [83]. FITC-or Texas red-conjugated secondary antibodies were used. Slides were mounted in VectaShield solution with DAPI (Vector Laboratories). Images were captured with an ORCA digital camera (Hamamatsu Photonics) on a Leica DM5500B microscope.

Whole-transcriptome RNA-seq analysis
Oocytes were collected from ovaries of 6-week-old wild-type or Ythdc1 fl/-Ddx4-Cre females by needle poking. Oocytes from Ythdc1 fl/-Ddx4-Cre females with gross abnormal morphology were excluded from studies. Total RNA was extracted from 25 oocytes per library using PicoPure RNA isolation kit with on-column genomic DNA digestion according to the manufacturer's instruction (Thermo Fisher Scientific). As a normalization control, each sample was spiked in with 0.2 pg synthesized Renilla luciferase mRNA before extraction. RNA-seq libraries were constructed by using Ovation RNA-seq system V2 (NuGEN) followed by Ovation Ultralow Library system (DR Multiplex System, NuGEN). Reverse transcription of total RNA was primed with a pool of primers that hybridize either to the 5' portion of the poly(A) sequence or randomly across the transcript. Per genotype, three biological replicate libraries were constructed. RNA-seq libraries were pooled and sequenced by three 150-bp paired-end runs on mid-output flow cells on the NextSeq 550 system (Illumina) (S1 Table). RNA-seq data are available under the NCBI/SRA number: SRP116737.

Differential expression analysis
Oocyte RNA-seq data were mapped using the RNA-Seq aligner STAR. The STAR genome was generated using the mouse mm10 genome assembly (Genome Reference GRCm38). STAR was run with the parameter-clip3pAdapterSeq GATCGGAAGAGCACACGTCTGAACTC-CAGTCAC. The SAM files from STAR were converted to BAM format using samtools view, reads were sorted by name using samtools sort, and separate lanes were merged into one file using samtools merge [84]. The number of reads in each genomic feature was quantified with HTSeq using the intersection-strict overlap setting [85]. Differential abundance between Ythdc1 fl/and Ythdc1-deficient oocytes was then analyzed using the R package DESeq2 on the HTSeq count files with default settings and an FDR cutoff of 0.01 [86].

Gene ontology (GO) analysis
Gene Ontology analysis was performed using the bioinformatics analysis resource database DAVID 6.8 [87]. Separate lists of differentially expressed up-regulated and down-regulated genes (all, FDR < 0.01, Fold change ! 2, and mean expression !100) were uploaded, with the Genbank_accession identifier selected and mus_musculus as the specified organism. A custom background list was supplied consisting of all genes with at least one read observed in any genotype or replicate in our RNA-seq libraries.

Bioinformatic analysis of local splicing variants (LSVs) by MAJIQ
Analysis of units of LSVs between Ythdc1 fl/and Ythdc1-deficient oocytes was performed using the MAJIQ software package [63]. MAJIQ v0.9.2 was run using the GRCm38 mm10 reference genome. Default settings were used for quantifying LSVs in the oocyte RNA-seq data and for the ΔPSI analysis. Please note that MAJIQ was not designed to identify alternative polyadenylation from RNA-seq data [63].

Bioinformatic analysis of alternative polyadenylation by ROAR
Alternative polyadenylation (APA) analysis was performed with the ROAR Bioconductor package in R [67]. The package's general workflow was followed. GV oocyte RNA-seq reads were mapped to the mm9 (NCBI37) genome using the RNA STAR aligner. ROAR was run using an annotation database of polyadenylation sites from the PolyADB version 2 [88]. The ratio of shorter to longer isoforms referred to as m/M ratio was computed for each sample using the counts of mapped reads and the lengths of the transcript's PRE and POST portions as defined using a multiple APA annotation file. The ratio of the m/M (WT) to the m/M (KO) yielded the ratio of a ratio (ROAR) values, which were used to identify shifts in polyadenylation site usage. 3' UTR lengthening or shortening was called when a Fisher test for all sample pairings returned nominal p-values < 0.05. We analyzed the RNA-seq data from the SRSF3 and SRSF7 knockdown experiments by Muller-McNicoll et al. [73] using the ROAR algorithm and reached the same conclusions on opposing changes on 3' UTR length, validating the ROAR algorithm.

Validation of differentially expressed genes by quantitative real-time PCR
Oocytes were collected from 6-week-old wild-type or Ythdc1 fl/-Ddx4-Cre or Ythdc1 fl/-Zp3-Cre females by needle poking of ovaries. Total RNA was extracted from oocytes using the Pico-Pure RNA Isolation Kit with on-column genomic DNA digestion (Thermo Fisher Scientific) and reverse transcribed by Superscript II reverse transcriptase (Invitrogen) using random hexamers. The resulting cDNA was quantified by real-time PCR on an ABI Prism 7000 thermocycler (Applied Biosystems) using Power SYBR Green Master Mix (Thermo Fisher Scientific).

Validation of local splicing variants and APA
For validation of local splicing variants, each PCR assay was optimized individually (S5 Table). The PCR cycles varied for each LSV, depending on transcript abundance. For APA validation, two pairs of PCR primers were designed: PRE and POST (before and after the polyadenylation site) (S5 Table). All assays used an amount of cDNA equivalent to one oocyte per PCR reaction. RT-PCR band quantification was performed using ImageJ.

Histological analysis
Testes and ovaries were prepared for histological analysis by fixation in Bouin's solution (Sigma Aldrich), followed by serial dehydration and paraffin infiltration and embedding. Serial sections were cut at 8 μm thickness, adhered to glass slides, and dried overnight. Slides were de-paraffinized with xylene and re-hydrated. Slides were then stained with hematoxylin, rinsed, and exposed to 0.1 M ammonia before staining with eosin. The slides were then dehydrated and mounted with Permount mounting media (Fisher Scientific). Images were taken on a DM5500B microscopy platform with a DFC450 camera (Leica Microsystems).

DNA constructs and mutagenesis
Wild-type mouse Ythdc1 coding region was amplified from bulk mouse testis cDNA samples by PCR. The double mutation (W377A, W428A) was introduced by PCR-based mutagenesis by mutating codons 377 (TGG) 428 (TGG) to 377 (GCG) 428 (GCG), resulting in W377A W428A amino acid changes. The entire coding region was cloned into the pcDNA3.1 vector for in vitro transcription.

In vitro transcription
Plasmids pcDNA3.1-wt-Ythdc1 and pcDNA3.1-Ythdc1-W377A W428A were verified by sequencing and linearized before in vitro transcription. Capped mRNAs were made by in vitro transcription with T7 mScript mRNA production System (CellSCRIPT) according to the manufacturer's instructions. Following in vitro transcription, template DNAs were digested by adding RNase-free DNase, and synthesized mRNA was purified by MEGAclear kit (Ambion).
A single mRNA band of the expected size was observed for each RNA sample on a 1% formaldehyde denaturing gel. Synthesized RNA was stored in aliquots at -80˚C.

Oocyte microinjection and LSV rescue assay
GV oocytes were collected from postnatal day 12 Ythdc1-deficient ovaries by enzymatic digestion (collagenase). Oocytes were microinjected with approximately 5 pl of wild-type or mutant Ythdc1 mRNA in water as previously described [89]. PND12 wild-type oocytes were mock injected with water as controls. Following microinjection, oocytes were returned to CZB medium with 2.5 μM milrinone and cultured overnight, followed by RNA extraction and reverse transcription. For LSV rescue experiments, each PCR assay was performed using an amount of cDNA equivalent to 0.5 oocyte. PCR bands were quantified using the Image J software. The rescue experiments were performed two times.

Transfection and co-immunoprecipitation
The FLAG-YTHDC1 expression construct was made by cloning the full-length mouse Ythdc1 coding sequence into a pcDNA6 vector containing a previously inserted 3xFLAG sequence 5' of the cloning site. V5 tagged constructs for Srsf7, Cstf1, Cstf2, Cpsf6, and Nudt21 were all produced by subcloning RT-PCR products amplified from bulk mouse testis cDNAs using the pcDNA 3.1/V5-His TOPO TA Expression Kit (Invitrogen, K4800). Srsf3 cDNA was cloned into a pcDNA6 vector containing a Myc tag 5' of the cloning site to express Myc-SRSF3. The HEK 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamine in a 37˚C humidified incubator at 5% CO 2 . Cells were co-transfected using Lipofectamine 2000 (Invitrogen) and cultured in Opti-mem media for 36 h. Transfected cells were harvested with RIPA buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 140 mM NaCl, 0.1% sodium deoxycholate) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by Dounce homogenization and solubilized by rocking at 4˚C for 30 min. Following centrifugation at 16,100 x g for 25 min, lysate supernatants were incubated with 1 mg/ml RNase A at room temperature for 30 min. Lysates were cleared by centrifugation at 16,100 x g for 20 min and incubated with Protein G agarose beads (Invitrogen, 15920010) for 1 h. After pre-clearing, either anti-FLAG (F-3165, Sigma), anti-V5 (R96025, ThermoFisher), or anti-Myc (631206, Clontech) was incubated with cell lysates rotating overnight at 4˚C. Equilibrated Protein G agarose beads were added to the lysates and incubated for 1 h. Immunoprecipitated protein complexes were washed three times with RIPA buffer supplemented with PMSF. Beads and respective input lysates were boiled with 2x SDS sample buffer for 5 min prior to SDS-PAGE and immunoblotting to nitrocellulose membranes.  Table. Differentially expressed genes between wild-type and Ythdc1-deficient oocytes from 6-week-old females. FDR cutoff of 0.01 was used. (XLSX) S3 Table. MAJIQ-identified local splicing variants (LSVs) between wild-type and Ythdc1deficient oocytes from 6-week-old females.