MicroRNAs (miRNAs) are a class of highly conserved small regulatory RNAs. After loading into Argonaute (AGO) proteins, miRNAs promote the degradation and reduced translation of the target mRNAs to which they bind. Consistent with the essential functions of miRNAs in development and physiology, elaborate mechanisms that control miRNA transcription and biogenesis have been uncovered. However, much less is known about the mechanisms that control miRNA turnover. Although miRNAs are generally very stable, with half-lives extending to days, some miRNAs are rapidly degraded. Thus, regulated miRNA decay mechanisms exist. Perhaps the most well-characterized pathway for miRNA turnover is a process called target-directed miRNA degradation (TDMD), which occurs when a miRNA engages a target with extensive complementarity. Unlike canonical targets, TDMD-inducing targets typically exhibit extended base pairing with the miRNA 3′ end. Multiple classes of targets that trigger TDMD have been identified, including synthetic transgenes, virally encoded transcripts, and endogenous protein-coding and noncoding RNAs. Nevertheless, the mechanism that leads to miRNA degradation when a TDMD target is encountered is poorly understood.

Nontemplated nucleotide addition and removal at the miRNA 3′ end occurs when a miRNA engages a TDMD target. This process, called tailing and trimming, has been proposed to be an essential step in the decay mechanism. However, depletion of terminal nucleotidyltransferases or nucleases that participate in tailing and trimming does not stabilize miRNAs undergoing TDMD. Thus, the role of tailing and trimming in this pathway remains unclear. Furthermore, trans-factors that are required for TDMD have yet to be identified. We performed a genome-wide CRISPR-Cas9 screen to investigate this important mechanism of miRNA regulation and to address these unresolved questions.

The highly conserved long noncoding RNA CYRANO triggers robust TDMD of miR-7 in the human cell line K562. An enhanced green fluorescent protein (EGFP) reporter transcript was designed to monitor decay of miR-7 in this system and used in a genome-wide CRISPR-Cas9 screen to identify factors that are required for TDMD. The screen revealed that TDMD is mediated by a cullin-RING ubiquitin ligase (CRL) complex containing the substrate adaptor ZSWIM8, as well as core CRL components elongins B and C (ELOB and ELOC), cullin 3 (CUL3), ARIH1, and RBX1. The ZSWIM8 ubiquitin ligase was required for TDMD induced by multiple transcripts in multiple cell lines. miRNA turnover mediated by the ZSWIM8 complex did not require tailing and trimming. Instead, ubiquitin transfer activity and activity of the proteasome was required for miRNA degradation directed by this complex. Moreover, the ZSWIM8 CRL interacted with AGO proteins. Conserved surface-exposed lysine residues on AGO2, particularly K493, were required for TDMD. These data suggest a role for direct ubiquitylation of AGO in this miRNA decay pathway. Small RNA sequencing in diverse cell lines after depletion of ZSWIM8 revealed stabilization of many miRNAs not previously known to be regulated by TDMD.

Base pairing of a miRNA with a TDMD-inducing transcript promotes broad structural rearrangements of AGO. Our findings suggest a model in which the ZSWIM8 ubiquitin ligase recognizes this TDMD-associated conformation, resulting in ubiquitylation and subsequent proteasomal decay of the miRNA-containing complex. This likely leads to concomitant release and …