| Summary: | MicroRNAs (miRNAs) are small, ~21 nucleotide RNAs that guide Argonaute (AGO) proteins to complementary target RNAs using base-pairing interactions. In canonical miRNA-mediated repression, AGO recruits repressive trans-acting factors to bound targets to promote their deadenylation, decapping, and subsequent decay. Although knowledge surrounding miRNA biogenesis and effector functions is extensive, the understanding of how miRNAs decay is still relatively nascent. Recent global measurements of miRNA stabilities in cells of mice and flies concluded that most miRNAs are very stable, with half-lives ranging from many hours to days. However, a minority of miRNAs were also observed to be labile, with half-lives of a few hours. Prior to our work, it was not known how this rapid turnover is specified and actuated, although a number of miRNA-degradation processes and associated effectors had been proposed. One particular pathway discovered in 2010, named “target-directed miRNA degradation” (TDMD), was shown to cause miRNA degradation in the presence of unusually highly complementary target RNAs, and held particular explanatory appeal. However, neither the mechanism nor the biological scope of this pathway, beyond a handful of examples, were definitively known.
We searched for factors involved in TDMD, and unexpectedly found that the ZSWIM8 Cullin-RING ligase was required for all known instances of TDMD that we tested. We then showed that, contrary to the prior model, TDMD proceeds by a mechanism where in the presence of a highly complementary target RNA, ZSWIM8 polyubiquitinates AGO and marks it for destruction, thereby exposing the normally protected miRNA to degradation. By perturbing ZSWIM8, we were able to confidently identify dozens of miRNAs likely undergoing targeted degradation in cells of flies, mice, and worms. Our data showed that TDMD was able to quantitatively explain the half-lives of most rapidly turned over miRNAs in cells of mice and flies, implying that this regulatory mode is pervasively used in bilaterian animals.
To interrogate the biological functions mediated by TDMD, we knocked out ZSWIM8 in mice and found that mutants died soon after birth with small body size, cyanotic appearance, and defects in heart and lung development. These aberrations were accompanied by accumulation of ~50 miRNAs across various tissues – in many cases resulting in significant repression of their targets. This work is consistent with the possibility that TDMD mediates important biological functions in animals, and further work will be needed to clarify the precise molecular and cellular bases of the phenotypes.
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