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. 2018 May 25;14(5):e1007412.
doi: 10.1371/journal.pgen.1007412. eCollection 2018 May.

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

Seth D Kasowitz  1 , Jun Ma  1   2 , Stephen J Anderson  2 , N Adrian Leu  1 , Yang Xu  1 , Brian D Gregory  2 , Richard M Schultz  2   3 , P Jeremy Wang  1
Affiliations

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

Seth D Kasowitz et al. PLoS Genet. .

Abstract

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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Expression and subcellular localization of YTHDC1 in oocytes and pre-implantation embryos.
(A) YTHDC1 expression in adult mouse tissues. ACTB and TUBB (β-tubulin) served as loading controls. Heart and skeletal muscle contain little ACTB. (B) Western blot analysis of YTHDC1 in oocytes and pre-implantation embryos. TUBB served as a loading control. Note the lower levels of YTHDC1 in GV-stage oocytes, when normalized to TUBB. (C) Localization of YTHDC1 in oocytes and pre-implantation embryos. DNA was stained with Sytox green. Abbreviations: P5, P12: postnatal days 5, 12; GV, germinal vesicle stage; MII, metaphase II; 1C, 2C, 4C: 1-cell, 2-cell, 4-cell embryos; M/B, morula/blastocyst.
Fig 2
Fig 2. Postnatal loss of male germ cells in Ythdc1fl/- Ddx4-Cre males.
Histological analysis of testes from Ythdc1 wild-type or heterozygous (left) and Ythdc1fl/- Ddx4-Cre (right) males at birth (postnatal day 0) (A), PND8 (B), PND25 (C), and 8 weeks (D). Arrows in panels A and B indicate prospermatogonia and spermatogonia respectively. Scale bars, 50 μm.
Fig 3
Fig 3. YTHDC1 is required for oocyte growth.
(A) Timeline of disruption of Ythdc1 in oocytes using Ddx4-Cre or Zp3-Cre. (B) Histological analysis of ovaries from 8-week-old wild-type and Ythdc1fl/- Ddx4-Cre mice. Inset, enlarged view of the primary follicle marked by white arrow. Scale bars, 100 μm. (C) Histological analysis of ovaries from 8-week-old wild-type and Ythdc1fl/- Zp3-Cre mice. Scale bars, 100 μm.
Fig 4
Fig 4. RNA-containing cytoplasmic granules in Ythdc1-deficient oocytes.
(A) Presence of large cytoplasmic granules in oocytes from 11-week-old Ythdc1fl/- Ddx4-Cre (cKO) females. (B) Cytoplasmic granules in oocytes from 11-week-old Ythdc1fl/- Ddx4-Cre females contain RNA. Nuclei/nuclear DNA and cytoplasmic RNA granules are marked by arrowheads (blue) and arrows (white), respectively. DAPI stains DNA only. Sytox green stains both DNA and RNA.
Fig 5
Fig 5. Changes in the splicing landscape in Ythdc1-deficient oocytes.
Oocytes were collected from 6-week-old Ythdc1fl/+ and Ythdc1fl/- Ddx4-Cre females. (A) Summary of local splicing variants (LSVs) identified by MAJIQ. Significant LSVs: ΔPSI (difference in percentage spliced in) > 0.2 and q < 0.05. (B) PCR validation and gene track view of one exon-skipping LSV in Tmem2. (C) PCR validation of LSVs affecting internal exons. Exons are represented as rectangles but not in scale. Skipped or retained exons are shown in red. Triangles denote the positions of PCR primers. Each PCR assays was performed three times using different samples.
Fig 6
Fig 6. Local splicing variants involve intron retention and 3’ UTR.
Oocytes were collected from 6-week-old Ythdc1fl/+ and Ythdc1fl/- Ddx4-Cre females. (A) PCR validation of intron-retaining LSVs. Introns and exons are represented as thick lines and rectangles, respectively. (B) Gene track view of a retained intron in the Dnpep Gene. Arrow indicates the affected intron. (C) PCR validation of LSVs in 3’ UTRs. The affected portion of the respective 3’ UTR is shown in red. Triangles denote the positions of PCR primers (A and C). (D) Gene track view of splicing variant in the Ifnar1 3’ UTR. The square bracket demarcates the spliced region of 3’ UTR in Ythdc1-deficient oocytes. Polyadenylation sites (PAS) are marked by vertical orange lines.
Fig 7
Fig 7. Alternative polyadenylation in Ythdc1-deficient oocytes.
Oocytes were collected from 6-week-old Ythdc1fl/+ and Ythdc1fl/- Ddx4-Cre females. (A) Pairwise comparison of PAS usage in wild-type and Ythdc1-deficient oocytes. PAS pairs with p<0.05 are shown in red or blue. (B) Gene track view and RT-PCR validation of alternative polyadenylation in Arl5a. Polyadenylation sites (PAS) are marked by vertical yellow lines. Exons and introns are marked by black bars and dotted lines, respectively, and the location of PRE and POST PCR fragments is shown. (C) RT-PCR validation results of alternative polyadenylation in 7 genes. Actb served as a loading control. (D) Quantification of RT-PCR products of 8 genes shown in panels A and B. A ratio [(PREKO/POSTKO)/(PREWT/POSTWT)] less than 1 indicates a higher level of the long isoform (with a longer 3’ UTR) in Ythdc1-deficient oocytes. A ratio of 1 for Frs2 indicates no preference between wild-type and mutant. Y-axis: mean ± SD. The experiments were performed in triplicates.
Fig 8
Fig 8. m6A-dependent rescue of alternative splicing defects in Ythdc1-deficient oocytes.
Postnatal day 12 Ythdc1fl/- Ddx4-Cre (cKO) oocytes were injected with mRNAs encoding wild-type or m6A-binding-deficient mutant (W377A W428A) YTHDC1 as marked on top of the gel panel, followed by RT-PCR analysis of LSVs. Left panel, schematic illustration of alternative splicing events for each transcript that correspond to the PCR products shown in the center panel. Each rectangle represents one exon, and exons subject to alternative splicing are marked red. Right panel, plot depicting quantification of ratios of band intensity or a single band intensity, with the value for wild-type oocyte (lane 1) set at 1. Asterisks indicate bands used for quantification. Enpp5 and Parp6: ratio of the upper band / the lower band; Tmem2: ratio of the lower band / the upper band. Actb serves as a loading control.
Fig 9
Fig 9. Association of YTHDC1 with pre-mRNA 3’end processing factors.
Recombinant proteins were expressed in HEK 293T cells. Co-immunoprecipitation was carried out in the presence of RNase. (A) Co-IP analysis of YTHDC1 with CPSF6 and NUDT21. * indicates antibody light chain. (B) Co-IP analysis of YTHDC1 with SRSF3 and SRSF7. ** indicates a non-specific band.
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