YTHDC2 control of gametogenesis requires helicase activity but not m⁶A binding

Abstract

Mechanisms regulating meiotic progression in mammals are poorly understood. The N6-methyladenosine (m6A) reader and 3′ → 5′ RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3′ UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.

RNA binding protein RBM46 regulates mitotic-to-meiotic transition in spermatogenesis

“…YTHDC2 predominantly targets the 3'UTRs (Fig. 6A) (30,31), coinciding with the distribution pattern of RBM46 along the length of mRNA. Analysis of YTHDC2 CLIP-seq identified 3764 and 21,313 YTHDC2-binding sites in P10 and adult mouse testis (31), respectively.…”

“…6A) (30,31), coinciding with the distribution pattern of RBM46 along the length of mRNA. Analysis of YTHDC2 CLIP-seq identified 3764 and 21,313 YTHDC2-binding sites in P10 and adult mouse testis (31), respectively. Among 511 and 398 YTHDC2 top targets in P10 and adult mouse testis listed in eCLIP mRNA targets substantially overlapped with YTHDC2 mRNA targets by CLIP-seq in P10 testis.…”

“…S8B). To precisely evaluate a possible coordinated role on their target mRNAs, we compared RBM46-binding sites by eCLIP-seq with YTHDC2 targets by individual-nucleotide resolution CLIP-seq (iCLIP-seq) from FLAG-tagged Ythdc2 knock-in mice at P14 ( 30 ) and a recently published YTHDC2 CLIP-seq in WT using YTHDC2 antibodies ( 31 ). YTHDC2 predominantly targets the 3′UTRs (Fig.…”

“…Recombinant human YTHDC2 protein has 3′→5' RNA helicase activity because it is an RNA-dependent adenosine triphosphatase (ATPase) that unwinds the RNA duplex with a 3′ overhang (26,27). Unexpectedly, recent genetic evidence demonstrated that m 6 A-binding capacity of YTHDC2 is not required for spermatogenesis (30,31). The loss of YTHDC2 3′→5' RNA helicase activity by a point mutation in the ATPase motif causes infertility with defects in meiotic prophase I progression (30,31).…”

“…Unexpectedly, recent genetic evidence demonstrated that m 6 A-binding capacity of YTHDC2 is not required for spermatogenesis (30,31). The loss of YTHDC2 3′→5' RNA helicase activity by a point mutation in the ATPase motif causes infertility with defects in meiotic prophase I progression (30,31). Male germ cells mutant for Ythdc2 initiate meiosis but fail to maintain prolonged meiotic prophase I with a premature exit into aberrant metaphase (26)(27)(28)32).…”