An unconventional YTH domain protein with essential functions

YTHDC2 is the largest member of the protein family containing YTH domains. These are RNA binding domains, most of which are m6A readers, the most frequent internal mRNA modification. Besides the YTH domain, the YTHDC2 mouse protein contains R3H, OB, RecA and Ankyrin repeat domains. Loss of YTHDC2 leads to infertile mice of both genders. The Pillai lab (University of Geneva) continued researching YTHDC2 and its function in fertility by investigating its domains' functional roles as well as its RNA and protein binding partners.

A CLIP experiment with testis tissue revealed over 30'000 YTHDC2 binding sites in thousands of transcripts. Over 90% of transcripts were mRNAs, and more than half of the sites were in the 3'UTR. The cross-linked sites contained U-rich motifs, and an examination of up- and downstream sequences did not discover an enrichment of the m6A methylation consensus motif.

These results and the weaker affinity to m6A RNA of the protein's YTH domain compared to those from other proteins prompted the question whether m6A binding is essential for YTHDC2's function in the germline. To investigate this, the researchers created knock-in mice in which a critical YTH domain residue for m6A binding was mutated. Mice homo- and heterozygous for this mutation were found to be viable and fertile and did not show any observable phenotype, especially in germline tissues. Matching these results is that the Drosophila YTHDC2 homolog is important for germline function but lacks a YTH domain.

Continuing with mouse genetics, the Pillai lab created mice carrying a YTHDC2 allele which contained a mutation rendering the helicase domain catalytically dead. This allele showed a dominant infertility phenotype as heterozygous cat-dead male mice failed to produce progeny when crossed with wild-type females. In addition, testicular transcriptome analysis of such a mutant revealed the downregulation of genes highly expressed in the meiotic stage of spermatogenesis.

To further study the transcriptome, the researchers applied single-cell sequencing to testicular germ cells from YTHDC2 knockout mice. They found that mutant germ cells contained a mixed transcriptome having at the same time mitosis- and meiosis-specific transcripts. "Single cell sequencing allowed the distinction of cells with a mixed identity from mitotic and meiotic cells," comments Kyrylo Krasnykov, co-first author of the study published in Molecular Cell. This revealed that Ythdc2 mutant germ cells start the transition from mitosis to meiosis but then get stuck and cannot complete it in the absence of YTHDC2, leading to the observed infertility phenotype.

The helicase's essential function for fertility warranted further investigation. Interestingly, the RecA helicase domain of YTHDC2 is split into two parts with two Ankyrin repeats located between them. These repeats are responsible for YTHDC2's interaction with the exoribonuclease XRN1. Studies conducted by Lingyun Li, the other co-first author of the study, showed that YTHDC2 has a low RNA unwinding activity in vitro, while the mutant protein lacking the ankyrin repeats showed increased activity. This helicase-breaking function of the repeats could be released by adding recombinant XRN1, which acts as a helicase accelerator.

Overall, the researchers favor a model in which YTHDC2 is essential to degrade mitotic transcripts for cells to undergo meiosis subsequently. "For the transition from the mitotic to the meiotic state, not only a transcriptional switch is needed, but the transcriptome needs to be cleared of remaining mitotic transcripts. YTHDC2 is responsible for this transcriptome clearance, and this involves not a couple of transcripts but entire sets. We are now focused on understanding how the RNA helicase activity contributes to this process," says Ramesh Pillai, last author of the study.  


Li, Krasnykov et al. (2022) Molecular Cell 82(9), 1678-1690.e12 (Open Access)

Image Legend. Graphical abstract from Li, Krasnykov et al. (2022) Molecular Cell published under a CC BY 4.0 licence.

Text: Dominik Theler