MicroRNAs (miRNAs) are short RNAs that direct widespread gene repression in the cells of humans and other animals. Each miRNA associates with an Argonaute (AGO) protein to form a silencing complex in which the miRNA pairs to sites within target mRNAs, and AGO recruits machinery that causes destabilization or reduced translation of the targeted transcript. In the aggregate, miRNAs reduce the output of most human genes, and most of the 90 broadly conserved miRNA families found in mammals are required for viability or proper development.

Most miRNAs are quite long-lived because association with AGO protects them from cellular nucleases. However, some miRNAs are relatively unstable. A potential explanation for the instability of these miRNAs comes from the observation that interactions with certain target sites can promote miRNA destruction, thereby inverting the typical regulatory logic. This phenomenon of target-directed miRNA degradation (TDMD) is exploited by some viruses, which produce transcripts that trigger the decay of specific host miRNAs that would otherwise impede viral replication. In addition, four endogenous transcripts have recently been found to trigger TDMD. These include the CYRANO long noncoding RNA, which directs efficient degradation of miR-7. However, the extent to which this phenomenon might accelerate the degradation of other miRNAs has been unclear, in part because proteins required for TDMD had not been identified.

The unusual target sites that trigger TDMD differ from the typical sites that mediate gene repression in that they not only pair to the 5′ region of the miRNA but also pair extensively to the miRNA 3′ region. This extensive pairing to the 3′ region can cause conformational changes that expose the miRNA 3′ terminus to enzymes that append or remove nucleotides—processes called tailing and trimming, respectively. The tailing and trimming observed in the presence of sites that trigger TDMD have been proposed to be obligate steps of the TDMD pathway. However, loss of an enzyme responsible for target-directed tailing has little influence on CYRANO-directed degradation of miR-7, which suggests that TDMD might occur through another mechanism. To learn more about this mechanism, we carried out a CRISPRi screen designed to identify proteins required for CYRANO-directed degradation of miR-7 in cultured human cells.

Our screen revealed that the ZSWIM8 protein was required for CYRANO-directed miR-7 degradation. ZSWIM8 was also required for other known examples of TDMD, including degradation triggered by a viral noncoding RNA and a cellular mRNA. Moreover, identification of miRNAs that increased after knocking out ZSWIM8 in different types of mouse and human cells implicated dozens of additional miRNAs as substrates of endogenous TDMD. Indeed, for cells in which miRNA half-lives were known, TDMD explained the destabilization of most short-lived miRNAs. Similar experiments that examined the consequences of knocking out the ZSWIM8 ortholog in Drosophila cells and nematodes indicated that endogenous TDMD also shapes miRNA levels and dynamics in invertebrate species.

ZSWIM8 is the substrate receptor of a Cullin-RING E3 ubiquitin ligase; this suggests an alternative model for TDMD that does not depend on miRNA tailing and trimming. In this model, the ZSWIM8 ubiquitin ligase recognizes the conformational changes that occur upon extensive pairing to the miRNA 3′ region, which leads to polyubiquitination of AGO. Polyubiquitinated AGO is then degraded by the 26S proteasome, thereby exposing …