The CNOT4 Subunit of the CCR4‐NOT Complex is Involved in mRNA Degradation, Efficient DNA Damage Repair, and XY Chromosome Crossover during Male Germ Cell Meiosis

Abstract

The CCR4-NOT complex is a major mRNA deadenylase in eukaryotes, comprising the catalytic subunits CNOT6/6L and CNOT7/8, as well as CNOT4, a regulatory subunit with previously undetermined functions. These subunits have been hypothesized to play synergistic biochemical functions during development. Cnot7 knockout male mice have been reported to be infertile. In this study, viable Cnot6/6l double knockout mice are constructed, and the males are fertile. These results indicate that CNOT7 has CNOT6/6L-independent functions in vivo. It is also demonstrated that CNOT4 is required for post-implantation embryo development and meiosis progression during spermatogenesis. Conditional knockout of Cnot4 in male germ cells leads to defective DNA damage repair and homologous crossover between X and Y chromosomes. CNOT4 functions as a previously unrecognized mRNA adaptor of CCR4-NOT by targeting mRNAs to CNOT7 for deadenylation of poly(A) tails, thereby mediating the degradation of a subset of transcripts from the zygotene to pachytene stage. The mRNA removal promoted by the CNOT4-regulated CCR4-NOT complex during the zygotene-to-pachytene transition is crucial for the appropriate expression of genes involved in the subsequent events of spermatogenesis, normal DNA double-strand break repair during meiosis, efficient crossover between X and Y chromosomes, and ultimately, male fertility.

RAD51AP2 is required for efficient meiotic recombination between X and Y chromosomes

“…To our knowledge, RAD51AP2 is the first protein that specifically participates in PAR DSB repair for ensuing XY crossover formation but is dispensable for autosomal crossover formation. Several genes have been reported to increase precocious XY separation during meiosis due to defects in DSB repair after disruption in mice (19,22,(45)(46)(47)(48)(49). It should be noted that all these genes, when mutated in mice, not only increased the frequency of XY separation but also impaired autosomal chromosome behaviors.…”

Chromodomain helicase DNA-binding domain 2 maintains spermatogonial self-renewal by promoting chromatin accessibility and mRNA stability

“… 34 It has been reported that from mid-pachytene to diplotene, DSB repair is mainly mediated by nonhomologous end joining (NHEJ) and comprises Ku70 and XRCC4 to repair DSB on the autosomes and 53BP1 to repair DSB on the XY chromosomes. 35 , 36 In meiosis I spermatocytes, the phosphorylated form of histone variant H2AX (γH2AX) marks sites of DNA damage throughout the nucleus, and as DNA is repaired, it remains on the sex chromosomes. 37 Because CHD2 is highly expressed in meiosis I spermatocytes and previous studies have shown that CHD2 is involved in NHEJ repair to promote DSB repair, 28 we wanted to know whether Chd2 haploinsufficiency would cause impaired DNA damage repair during mouse spermatogenesis.…”

Unearthing of key genes driving the infertility risk of cryptorchidism via weighted gene co- expression network

“…Further studies are required to clarify the exact function of these genes in cryptorchidism-related infertility, and whether they could become candidate drug targets to prevent and treat cryptorchidism-related infertility. Yes [34,35] The CNOT4 subunit of the CCR4-NOT complex is crucial in male germ cell meiosis mediating multiple important processes such as XY chromosome crossover [34]. CAF1 (part of the CCR4-NOT complex) was essential for spermatogenesis [35].…”

“…To seek the association of hub genes with cryptorchidism-related infertility, we retrieved the published literature for possible proof. Though no hub gene was found exactly involved in cryptorchidism-related infertility, ten genes (CCR4, LMNB1, LMNB1, MTNR1B, TAC3, TRHR, TRHR, RAD54L, CCNA1, SMC1B, GPRC6A, SYCE2) played a key role in infertility or spermatogenesis [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50], indicating that these hub genes might be involved in cryptorchidism-related infertility. Additionally, there was no direct evidence of the function of CHRM4 and TAS2R19 in infertility.…”