The
mir-35 family of microRNAs (miRNA) in Caenorhabditis elegans are maternally contributed as well as zygotically expressed in early embryos. Expression of the
mir-35 family, which consists of 8 miRNAs,
mir-35-42, is essential for viability; complete loss of the
mir-35 family results in embryonic lethality.
mir-35 family abundance is developmentally regulated and sharply decays at the end of embryogenesis. While there is much known about the biogenesis and functions of miRNAs, very little is known about the decay mechanisms of miRNAs. Because of this tight regulation of the
mir-35 family during development, this family of miRNAs is an interesting model for studying the mechanisms of miRNA decay. The
mir-35 family has two defining characteristics: the first is a shared, family-specific seed sequence (nucleotides 2-8 at the 5' of the miRNA), and the second is preferential loading into the Argonaute protein ALG-2 (rather than the better studied ALG-1). We are interested in ascertaining whether either of these characteristics play a role in the regulated decay of the
mir-35 family. To examine if the
mir-35 family turnover 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 found that the
mir-35 seed mutants were loaded into ALG-2 similarly to wild type
mir-35, indicating that altered Argonaute loading is not the cause of the altered turnover of the
mir-35 seed mutants. Therefore, ALG-2 loading is not sufficient for regulated turnover since mutant
mir-35 is misregulated despite its normal loading into ALG-2. Having shown that the seed sequence is necessary for
mir-35 decay, we next interrogated whether the seed is sufficient for the regulated decay of the
mir-35 family. We mutated all residues of
mir-35 outside of the seed region and observed that these miRNAs are decayed fairly similarly to wild type
mir-35. Thus, the seed is largely sufficient to elicit developmentally timed decay. Overall, our findings contribute to a better understanding of the regulation of
mir-35 family abundance in early development. By elucidating the mechanisms of
mir-35 family decay, this research will offer insight into the broader mechanisms that couple miRNA decay to developmental progression.