To kill a microRNA: emerging concepts in target-directed microRNA degradation

Abstract

MicroRNAs (miRNAs) guide Argonaute (AGO) proteins to bind mRNA targets. Although most targets are destabilized by miRNA-AGO binding, some targets induce degradation of the miRNA instead. These special targets are also referred to as trigger RNAs. All triggers identified thus far have binding sites with greater complementarity to the miRNA than typical target sites. Target-directed miRNA degradation (TDMD) occurs when trigger RNAs bind the miRNA-AGO complex and recruit the ZSWIM8 E3 ubiquitin ligase, leading to AGO ubiquitination and proteolysis and subsequent miRNA destruction. More than 100 different miRNAs are regulated by ZSWIM8 in bilaterian animals, and hundreds of trigger RNAs have been predicted computationally. Disruption of individual trigger RNAs or ZSWIM8 has uncovered important developmental and physiologic roles for TDMD across a variety of model organisms and cell types. In this review, we highlight recent progress in understanding the mechanistic basis and functions of TDMD, describe common features of trigger RNAs, outline best practices for validating trigger RNAs, and discuss outstanding questions in the field.

Graphical Abstract

Figure 1.

The miRNA life cycle. (A) miRNA biogenesis and targeting are depicted. After sequential processing in the nucleus and cytosol by DROSHA/Microprocessor and DICER, respectively, the miRNA duplex is loaded into the AGO effector protein and one strand is removed. The fate of the AGO–miRNA complex and target RNA depend on the extent of base-pairing between miRNA and target. These outcomes include target degradation via deadenylation and decapping, target degradation via endonucleolytic cleavage (also known as slicing), and miRNA degradation. (B) In the current model of target-directed miRNA degradation, extensive complementarity between the AGO–miRNA complex and target RNA induces a stabilized ternary complex (1), which recruits the ZSWIM8 substrate adapter, other subunits of the E3 ubiquitin ligase including neddylated (N) CUL3, ELONGINS B and C, ARIH1, and RBX1, and an ubiquitin-conjugating enzyme (E2) (2), leading to polyubiquitination (U) (3) and proteasomal degradation of AGO (4), followed by degradation of the deprotected miRNA (5). No longer bound to miRNA, the target RNA can interact with a new AGO–miRNA complex (6), initiating another round of miRNA degradation.

Figure 2.

ZSWIM8 protein structure and conservation. (A) Linear diagram of the ZSWIM8 protein showing the size and location of the BC box (orange), CUL box (green), and SWIM domain (teal), as well as several regions of predicted disorder (coral). Below, conservation scores (gray) derived from CONSURF-DB (105) and disorder scores (coral) derived from IUPRED (104) have been plotted along the length of ZSWIM8. In general, intrinsically disordered regions are poorly conserved. (B) The AlphaFold prediction (AF-A7E2V4-F1) of human ZSWIM8 protein is shown (107,108). The BC box (orange), CUL box (green), and SWIM domain (teal) are tightly clustered at one end of the protein and accessible to solvent and other proteins. The conserved protein core (gray) consists of repeating alpha helices that form a solenoid-like structure. The disordered regions (coral) are not confidently predicted. (C) AlphaFold-Multimer prediction (via COSMIC) for ZSWIM8 in complex with ELOB (purple), ELOC (navy), and CUL3 (light blue)(109). The inset image provides another look at the ELOB–CUL3–ZSWIM8 interface from a different angle.

Figure 3.

Structures of AGO–miRNA–target and AGO–miRNA–trigger complexes. (A) The structure of human AGO2 (PDB 6N4O, cartoon plus surface representation generated with UCSF Chimera (106)) loaded with miR-122 and bound to a target RNA (black) via seed and 4 nts of supplemental pairing (60). The 5′ end of the miRNA is protected by the MID domain (pink) and the 3′ end of the miRNA (asterisk) is protected by the PAZ domain (blue). The central region of the miRNA is unpaired and the corresponding target nts are not visible in this structure. (B) The structure of human AGO2 (PDB 6NIT, cartoon plus surface representation generated with UCSF Chimera) loaded with miR-122 and bound to a TDMD site (bu2) with 10 nts of 3′ pairing (black) (15). The 5′ end of the miRNA is still protected by the MID domain (pink), however, the 3′ end of the miRNA (asterisk) is no longer protected by the PAZ domain (blue). The 3′ half of the miRNA has rotated, and the sugar-phosphate backbone is no longer intimately associated with AGO. A similar structure was solved with AGO2, miR-27a, and the HSUR1 TDMD site (15).

Figure 4.

Experimental strategies to validate candidate trigger RNAs. (A) The most common approach for interrogating the activity of a TDMD site is depicted. The trigger RNA or a part thereof is inserted in the 3′ UTR of a reporter gene and transiently overexpressed. Here, the expectation is that the wildtype TDMD site should decrease the miRNA, whereas a mutated TDMD site should have no effect. The opposite strand of the miRNA duplex (miR^os) should not be affected. In ZSWIM8 KO cells, the miRNA will be increased if the endogenous trigger is normally expressed and overexpressing the wildtype TDMD site should not affect the miRNA. Fold changes are compared to WT cells expressing only the coding sequence (CDS). (B) The recommended approach for interrogating the activity of a TDMD site is depicted. CRISPR-Cas9 is used to disrupt the endogenous TDMD site, which should lead to an increase of the miRNA in wildtype (WT) cells. As above, the opposite strand of the miRNA duplex should not be affected. In ZSWIM8 knockout (KO) cells, the miRNA is already increased and disrupting the TDMD site should not have any additional effect. Fold changes are compared to WT cells expressing a control sgRNA.

Figure 5.

Potential advantages of regulating miRNA expression via TDMD. (A) TDMD can deplete select miRNAs produced from a miRNA cluster. (B) TDMD can globally suppress expression of miRNAs expressed by paralogous genes at distant loci (chromosome map created with BioRender.com). (C) Coupled with transcription (signified by gray on/off arrows), TDMD can sharpen the dynamic expression of the miRNA, thus enabling the miRNA to reach a new steady state level more quickly.