The Rapidly Evolving X-linked miR-506 Family Finetunes Spermatogenesis to Enhance Sperm Competition

Reviewer #1 (Public Review):

Wang et al investigated the evolution, expression, and function of the X-linked miR-506 miRNA family. They showed that the miR-506 family underwent rapid evolution. They provided evidence that miR-506 appeared to have originated from the MER91C DNA transposons. Human MER91C transposon produced mature miRNAs when expressed in cultured cells. A series of mouse mutants lacking individual clusters, a combination of clusters, and the entire X-linked cluster (all 22 miRNAs) were generated and characterized. The mutant mice lacking four or more miRNA clusters showed reduced reproductive fitness (litter size reduction). They further showed that the sperm from these mutants were less competitive in polyandrous mating tests. RNA-seq revealed the impact of deletion of miR-506 on the testicular transcriptome. Bioinformatic analysis analyzed the relationship among miR-506 binding, transcriptomic changes, and target sequence conservation. The miR-506-deficient mice did not have apparent effect on sperm production, motility, and morphology. Lack of severe phenotypes is typical for miRNA mutants in other species as well. However, the miR-506-deficient males did exhibit reduced litter size, such an effect would have been quite significant in an evolutionary time scale. The number of mouse mutants and sequencing analysis represent a tour de force. This study is a comprehensive investigation of the X-linked miR-506 miRNA family. It provides important insights into the evolution and function of the miR-506 family.

The conclusions of this preprint are mostly supported by the data except being noted below. Some descriptions need to be revised for accuracy.

L219-L285: The conclusion that X-linked miR-506 family miRNAs are expanded via LINE1 retrotransposition is not supported by the data. LINE1s and SINEs are very abundant, accounting for nearly 30% of the genome. In addition, the LINE1 content of the mammalian X chromosome is twice that of the autosomes. One can easily find flanking LINE1/SINE repeat. Therefore, the analyses in Fig. 2G, Fig. 2H and Fig. S3 are not informative. In order to claim LINE1-mediated retrotransposition, it is necessary to show the hallmarks of LINE1 retrotransposition, which are only possible for new insertions. The X chromosome is known to be enriched for testis-specific multi-copy genes that are expressed in round spermatids (PMID: 18454149). The conclusion on the LINE1-mediated expansion of miR-506 family on the X chromosome is not supported by the data and does not add additional insights. I think that the LINE1 related figure panels and description (L219-L285) need to be deleted. In discussion (L557-558), "...and subsequently underwent sequence divergence via LINE1-mediated retrotransposition during evolution" should also be deleted. This section (L219-L285) needs to deal only with the origin of miR-506 from MER91C DNA transposons, which is both convincing and informative.

Fig. 3A: can you speculate/discuss why the miR-506 expression in sperm is higher than in round spermatids?

Reviewer #2 (Public Review):

In this paper, Wang and collaborators characterize the rapid evolution of the X-linked miR-506 cluster in mammals and characterize the functional reference of depleting a few or most of the miRNAs in the cluster. The authors show that the cluster originated from the MER91C DNA transposon and provide some evidence that it might have expanded through the retrotransposition of adjacent LINE1s. Although the animals depleted of most miRNAs in the cluster show normal sperm parameters, the authors observed a small but significant reduction in litter size. The authors then speculate that the depletion of most miRNAs in the cluster could impair sperm competitiveness in polyandrous mating. Using a successive mating protocol, they show that, indeed, sperm lacking most X-linked miR-506 family members is outcompeted by wild-type sperm. The authors then analyze the evolution of the miR-506 cluster and its predicted targets. They conclude that the main difference between mice and humans is the expansion of the number of target sites per transcript in humans.

The conclusions of the paper are, in most cases, supported by the data; however, a more precise and in-depth analysis would have helped build a more convincing argument in most cases.

1. In the abstracts and throughout the manuscript, the authors claim that "... these X-linked miRNA-506 family miRNA [...] have gained more targets [...] " while comparing the human miRNA-506 family to the mouse. An alternative possibility is that the mouse has lost some targets. A proper analysis would entail determining the number of targets in the mouse and human common ancestor.

2. The authors claim that the miRNA cluster expanded through L1 retrotransposition. However, the possibility of an early expansion of the cluster before the divergence of the species while the MER91C DNA transposon was active was not evaluated. Although L1 likely contributed to the diversity within mammals, the generalization may not apply to all species. For example, SINEs are closer on average than L1s to the miRNAs in the SmiR subcluster in humans and dogs, and the horse SmiR subcluster seems to have expanded by a TE-independent mechanism.

3. Some results are difficult to reconcile and would have benefited from further discussion. The miR-465 sKO has over two thousand differentially expressed transcripts and no apparent phenotype. Also, the authors show a sharp downregulation of CRISP1 at the RNA and protein level in the mouse. However, most miRNAs of the cluster increase the expression of Crisp1 on a reporter assay. The only one with a negative impact has a very mild effect. miRNAs are typically associated with target repression; however, most of the miRNAs analyzed in this study activate transcript expression.

4. More information is required to interpret the results of the differential RNA targeting by the murine and human miRNA-506 family. The materials and methods section needs to explain how the authors select their putative targets. In the text, they mention the use of four different prediction programs. Are they considering all sites predicted by any method, all sites predicted simultaneously by all methods, or something in between? Also, what are they considering as a "shared target" between mice and humans? Is it a mRNA that any miR-506 family member is targeting? Is it a mRNA targeted by the same miRNA in both species? Does the targeting need to occur in the same position determined by aligning the different 3'UTRs?

5. The authors highlight the particular evolution of the cluster derived from a transposable element. Given the tendency of transposable elements to be expressed in germ cells, the family might have originated to repress the expression of the elements while still active but then remained to control the expression of the genes where the element had been inserted. The authors did not evaluate the expression of transcripts containing the transposable element or discuss this possibility. The authors proposed an expansion of the target sites in humans. However, whether this expansion was associated with the expansion of the TE in humans was not discussed either. Clarifying whether the transposable element was still active after the divergence of the mouse and human lineages would have been informative to address this outstanding issue.

Post-transcriptional regulation is exceptionally complex in male haploid cells, and the functional relevance of many regulatory pathways remains unclear. This manuscript, together with recent findings on the role of piRNA clusters, starts to clarify the nature of the selective pressure that shapes the evolution of small RNA pathways in the male germ line.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors conducted a comprehensive study of the X-linked miR-506 family miRNAs in mice on its origin, evolution, expression, and function. They demonstrate that the X-linked miR-506 family, predominantly expressed in the testis, may be derived from MER91C DNA transposons and further expanded by retrotransposition. By genetic deletion of different combinations of 5 major clusters of this miRNA family in mice, they found these miRNAs are not required for spermatogenesis. However, by further examination, the mutant mice show mild fertility problem and inferior sperm competitiveness. The authors conclude that the X-linked miR-506 miRNAs finetune spermatogenesis to enhance sperm competition.

Strengths:

This is a comprehensive study with extensive computational and genetic dissection of the X-linked miR-506 family providing a holistic view of its evolution and function in mice. The finding that this family miRNAs could enhance sperm competition is interesting and could explain their roles in finetuning germ cell gene expression to regulate reproductive fitness.

Weaknesses:

The authors specifically addressed the function of 5 clusters of X-link miR-506 family containing 19 miRNAs. There is another small cluster containing 3 miRNAs close to the Fmr1 locus. Would this small cluster act in concert with the 5 clusters to regulate spermatogenesis? In addition, any autosomal miR-506 like miRNAs may compensate for the loss of X-linked miR-506 family. These possibilities should be discussed.

Direct molecular link to sperm competitiveness defect remains unclear but is difficult to address.