INTERROGATING THE REGULATION OF THE MIR-35 FAMILY OF MICRORNAS IN C. ELEGANS

MicroRNA (miRNA) abundance is tightly controlled by regulation of biogenesis and decay. While there is much known about the biogenesis and functions of miRNAs, very little is known about the decay mechanisms of miRNAs. The mir-35 family of microRNAs (miRNA) in Caenorhabditis elegans are maternally contributed as well as zygotically expressed in early embryos. mir-35 family abundance is developmentally regulated and sharply decays at the end of embryogenesis. Because of this tight regulation of the mir-35 family, this family of miRNAs is an interesting model for studying the mechanisms of miRNA decay. To examine if the mir-35 family decay is seed sequence-specific, we mutated the seed sequence of the mir-35 miRNA via CRISPR and monitored levels of the mutant mir-35 as it compares to wild type mir-35. We detected perdurance of mutant mir-35 past embryogenesis, demonstrating the necessity of the seed sequence in regulating mir-35 family decay. We next mutated all residues of mir-35 outside of the seed region and observed that, although sequences outside the seed (i.e. the 3’ end) regulate mir-35 abundance in the embryo, they do not control decay at the embryo to larval transition. Modifications of the miRNA 3’ end by tailing and trimming are neither prevalent nor correlated with changes in decay, further suggesting that base pairing of the miRNA 3’ end is not a core feature of this mechanism. Thus, the seed is largely sufficient to elicit this developmentally-timed decay. Our findings demonstrate that seed sequence-specific decay can selectively and coherently regulate all redundant members of a miRNA seed family, a class of mechanism that has great biological and therapeutic potential for dynamic regulation of a miRNA family’s target repertoire