MicroRNAs control gene expression through binding to complementary target mRNAs. Although the roles of microRNAs in C. elegans larval development have been extensively studied, relatively little is understood about the function of microRNAs in embryonic development. The mir-35-41 microRNA cluster is expressed maternally and in embryos, and is essential for embryonic development and fecundity. Here, we show that the mir-35-41 microRNA cluster promotes hermaphrodite sex determination and dosage compensation via regulation of its target gene sup-26.We observed that many transcripts are abberantly upregulated in mir-35-41(nDf50) mutant embryos, but that these transcripts are not enriched for mir-35-41 target sites. We hypothesized that mir-35-41 might indirectly repress these transcripts by promoting the function of a transcriptional repressor. By analyzing publicly-available gene expression data, we found that the transcriptional changes observed in mir-35-41(nDf50) are highly similar to those observed in dosage compensation mutant embryos (sdc-2(lf)). Additionally, mir-35-41(nDf50) strongly enhances her-1(gf) masculinization, another hallmark of compromised dosage compensation. We sought a target gene that might link mir-35-41 to dosage compensation. SUP-26 is an RNA-binding protein which was cloned as a suppressor of her-1(gf) masculinization, and is a predicted mir-35-41 target gene. Sup-26(lf) is epistatic to mir-35-41(nDf50);her-1(gf) masculinization and to the derepression of dosage-compensated genes observed in mir-35-41(nDf50) embryos. Thus, the dosage compensation phenotypes of mir-35-41(nDf50) are dependent on SUP-26, supporting a role for SUP-26 downstream of mir-35-41 in regulating dosage compensation.Finally, we performed high-throughput sequencing-crosslinking immunoprecipitation (HITS-CLIP) to define the targets of SUP-26 binding. Through this method, we identified >800 RNA peaks of direct SUP-26 binding. Nearly all of the SUP-26-bound regions are in the 3'UTRs of protein coding genes (>94% of peaks). Among the targets of SUP-26 binding are members of the dosage compensation complex, providing a putative link between mir-35-41, SUP-26, and dosage compensation. Overall, this work provides surprising new insight into the role that microRNAs play in regulating embryonic gene expression, and draws the first link between microRNAs and dosage compensation. .

Affiliation:
- University of Massachusetts Medical School, Worcester, MA