Expression, not sequence, distinguishes miR-238 from its miR-239ab sister miRNAs in promoting longevity in Caenorhabditis elegans

MicroRNAs (miRNAs) regulate gene expression by base-pairing to target sequences in messenger RNAs (mRNAs) and recruiting factors that induce translational repression and mRNA decay. In animals, nucleotides 2-8 at the 5 end of the miRNA, called the seed region, are often necessary and sometimes sufficient for functional target interactions. MiRNAs that contain identical seed sequences are grouped into families where individual members have the potential to share targets and act redundantly. A rare exception seemed to be the miR-238/239ab family in Caenorhabditis elegans, as previous work indicated that loss of miR-238 reduced lifespan while deletion of the miR-239ab locus resulted in enhanced longevity and thermal stress resistance. Here, we re-examined these potentially opposing roles using new strains that individually disrupt each miRNA sister. We confirmed that loss of miR-238 is associated with a shortened lifespan but could detect no longevity or stress phenotypes in animals lacking miR-239a or miR-239b, individually or in combination. Additionally, dozens of genes were mis-regulated in miR-238 mutants but almost no gene expression changes were detected in either miR-239a or miR-239b mutants compared to wild type animals. We present evidence that the lack of redundancy between miR-238 and miR-239ab is independent of their sequence differences; miR-239a or miR-239b could substitute for the longevity role of miR-238 when expressed from the miR-238 locus. Altogether, these studies disqualify miR-239ab as negative regulators of aging and demonstrate that expression, not sequence, dictates the specific role of miR-238 in promoting longevity. Author SummaryMicroRNAs (miRNAs) are tiny non-coding RNAs that function in diverse biological pathways. To exert their regulatory influence, miRNAs bind to specific target RNAs through partial base-pairing. A critical aspect of this miRNA-target engagement is the seed sequence, nucleotides 2-8 of the miRNA. MiRNAs that share seed sequences are grouped into families and presumed to have similar functions. Yet, other factors, such as non-seed sequences in the miRNA and its expression level, can also contribute to target regulation and result in distinct roles for miRNAs within a family. To better understand how miRNA family members can have specific functions, we focused on miR-238 and its sisters, miR-239a and miR-239b, because these miRNAs had previously been reported to play opposing longevity roles in the nematode C. elegans. Using new genetic tools, we found that loss of miR-238 alone leads to the misregulation of many genes and a reduced lifespan. However, the lack of miR-239a, miR-239b, or both sisters had almost no effect on gene expression or longevity compared to wild type animals. Strikingly, though, miR-239a or miR-239b could substitute for the aging role of miR-238 when expressed from the miR-238 locus. Thus, expression, not sequence, is the predominant distinguishing feature of mir-238 that bestows upon it a role in aging not shared with the other family members.