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Functions of X-linked miR-506 family in Reproduction
Cell and Molecular Biology
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MicroRNAs (miRNAs) are ~22nt small non-coding RNAs that play a pivotal role in both development and adulthood physiology. Our lab has previously discovered that the miR-506 family, which consists of 22 miRNAs existing in 5 clusters expanding a ~62kb region near Slitrk2 and 1 cluster expanding a ~22kb region close to Fmr1 on the X chromosome in mice. Derived from the same ancestors, these miRNAs are preferentially expressed in the testis and have undergone rapid evolution with variable seed sequences, whereas the two protein-coding genes (Slitrk2 and Fmr1) flanking the 6 clusters remain highly conserved among all mammalian species. The quick evolution and testis preferential expression of these X-linked miRNAs strongly suggest that they are under selective pressure and play an important role in fine-tuning certain molecular events to ensure the production of high quality sperm. However, these hypotheses remain untested. My dissertation projects aimed to unveil the physiological roles of the miR-506 family miRNAs. Using the CRISPR-Cas-based genome editing technology, we deleted individual or 5 clusters of the miR-506 family in the mouse genome to analyzed potential phenotype. Interestingly, we found that ablation of one or two miRNAs, or even one cluster of several miRNAs did not generate any discernable defects in either spermatogenesis or male fertility. When we deleted four or five of the 6 miRNA clusters, we started to see some phenotype in these KO males, characterized by slightly reduced litter sizes and longer litter intervals despite normal testis weight and normal sperm counts. When the typical one male-one female mating scheme was used, the KO males were only subfertile. However, when a female was mated sequentially with either a wild type male first and a KO second, or a KO first followed by a WT male, no or much fewer pups were derived from the KO sperm, suggesting the KO sperm are less fit in fertilizing eggs. Indeed, in vitro fertilization assays showed that the KO sperm were less competitive in fertilizing wild type eggs. Our data suggest that sperm produced in the absence of these X-linked miRNAs are less fertile/competitive than wild-type sperm, suggesting that the miR-506 family miRNAs function to fine-tune certain molecular processes that render sperm better fitness. Among the miR-506 family, 6 miRNAs of the miR-465 cluster have similar seed sequences and are preferentially expressed in the testis. Surprisingly, loss of miR-465 cluster did not cause male fertility defects, but led to sex ratio bias among KO offspring, and the distorted sex ratio was found to result from female-biased lethality as early as embryonic day (E) 8.5. It turns out that miR-465 cluster miRNAs are also highly expressed in extra-embryonic tissues at E7.5. The female-biased lethality is likely due to dysregulation numerous target genes known critical for the survival of the female embryos, e.g., Alkbh1, a tRNA demethylation enzyme. Taken together, the data presented in this dissertation uncovered the physiological roles of the miR-506 family. Consistent with our previous reports, miRNAs tend to act as a group, and loss of function of one miRNA can be easily compensated by other members of the same cluster or the same family. Thus, inactivation of a single miRNA rarely leads to a discernable phenotype in mice. To reveal the true physiological role of miRNAs within one cluster or one family, simultaneous inactivation of most or all of the miRNAs is required.