The XRN1-regulated RNA helicase activity of YTHDC2 ensures mouse fertility independently of m6A recognition.

The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by "reader" proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3' UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3'→5' RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5'→3' exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.

Destabilization of mRNAs enhances competence to initiate meiosis in mouse spermatogenic cells.

The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and to promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we have used the enhanced resolution of scRNA-seq and bulk RNA-seq of developmentally synchronized spermatogenesis to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including the transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the transcriptional regulator STRA8-MEIOSIN, which is required for the meiotic G1/S phase transition and for meiotic gene expression. We conclude that, in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis.

Regulation of m6A Transcripts by the 3'→5' RNA Helicase YTHDC2 Is Essential for a Successful Meiotic Program in the Mammalian Germline.

N6-methyladenosine (m6A) is an essential internal RNA modification that is critical for gene expression control in most organisms. Proteins with a YTH domain recognize m6A marks and are mediators of molecular functions like RNA splicing, mRNA decay, and translation control. Here we demonstrate that YTH domain-containing 2 (YTHDC2) is an m6A reader that is essential for male and female fertility in mice. High-throughput mapping of the m6A transcriptome and expression analysis in the Yhtdc2 mutant testes reveal an upregulation of m6A-enriched transcripts. Our biochemical studies indicate that YTHDC2 is an RNA-induced ATPase with a 3'→5' RNA helicase activity. Furthermore, YTHDC2 recruits the 5'→3' exoribonuclease XRN1 via Ankyrin repeats that are inserted in between the RecA modules of the RNA helicase domain. Our studies reveal a role for YTHDC2 in modulating the levels of m6A-modified germline transcripts to maintain a gene expression program that is conducive for progression through meiosis.

Cryptic splice site poisoning and meiotic arrest caused by a homozygous frameshift mutation in RBMXL2: A case report.

Gene expression in meiotic cells in the testis is characterized by intense transcriptional activity and alternative splicing. These processes are mainly controlled by RNA-binding proteins expressed strongly in germ cells. Functional impairments in any of these proteins' functions can lead to defects in meiosis and thus severe male infertility. Here, we have identified a homozygous frameshift mutation (NM_014469.4:c.301dup; p.Ser101LysfsTer29) in the RNA-binding motif protein, X-linked like 2 (RBMXL2) gene in a man with an azoospermia due to meiotic arrest. As RBMXL2 is known to be crucial for safeguarding the meiotic transcriptome in mice testes, we hypothesized that this variant leads to cryptic splice site poisoning. To determine the variant's impact on spermatogenesis, we confirmed the absence of RBMXL2 protein in the patient's testis tissue and then evidenced abnormal expression of several spermatogenesis proteins (e.g. meiosis-specific with coiled-coil domain) known to be altered in rbmxl2 knock-out mice with meiotic arrest. Our results indicate that RBMXL2's function in spermatogenesis is conserved in mammals. We hypothesize that deleterious variant in the RBMXL2 gene can result in male infertility and complete meiotic arrest, due to the disruption of gene expression by cryptic splice site poisoning.

ketu mutant mice uncover an essential meiotic function for the ancient RNA helicase YTHDC2.

Mechanisms regulating mammalian meiotic progression are poorly understood. Here we identify mouse YTHDC2 as a critical component. A screen yielded a sterile mutant, 'ketu', caused by a Ythdc2 missense mutation. Mutant germ cells enter meiosis but proceed prematurely to aberrant metaphase and apoptosis, and display defects in transitioning from spermatogonial to meiotic gene expression programs. ketu phenocopies mutants lacking MEIOC, a YTHDC2 partner. Consistent with roles in post-transcriptional regulation, YTHDC2 is cytoplasmic, has 3'→5' RNA helicase activity in vitro, and has similarity within its YTH domain to an N6-methyladenosine recognition pocket. Orthologs are present throughout metazoans, but are diverged in nematodes and, more dramatically, Drosophilidae, where Bgcn is descended from a Ythdc2 gene duplication. We also uncover similarity between MEIOC and Bam, a Bgcn partner unique to schizophoran flies. We propose that regulation of gene expression by YTHDC2-MEIOC is an evolutionarily ancient strategy for controlling the germline transition into meiosis.