A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming

Abstract

MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to repress target messenger RNAs. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter highly complementary targets, which can trigger a process called target-directed miRNA degradation (TDMD). During TDMD, miRNAs undergo tailing and trimming, suggesting that this is an important step in the decay mechanism. We identified a cullin-RING ubiquitin ligase (CRL), containing the substrate adaptor ZSWIM8, that mediates TDMD. The ZSWIM8 CRL interacts with AGO proteins, promotes TDMD in a tailing and trimming-independent manner, and regulates miRNA expression in multiple cell types. These findings suggest a model in which the ZSWIM8 ubiquitin ligase mediates TDMD by directing proteasomal decay of miRNA-containing complexes engaged with highly complementary targets.

Fig. 1.. A genome-wide CRISPR-Cas9 screen for TDMD factors reveals a cullin-RING E3 ubiquitin ligase.

(A) Location of the miR-7 binding site in CYRANO (also known as OIP5-AS). UCSC Genome Browser PhyloP and PhastCons tracks shown (hg38). (B) Loss of CYRANO in the K562 EGFP^miR−7 reporter cell line results in reduced fluorescence, as shown by flow cytometry (left), and increased miR-7 abundance, as shown by northern blotting (right). (C) Overview of the CRISPR-Cas9 screen. (D) Genes plotted by MAGeCK rank in screens performed in CYRANO^+/+ and CYRANO^−/− EGFP^miR−7 cells. Screens were conducted in two independent clones per genotype. CRL components (red), NEDDylation factors (blue), and known miR-7 regulators (green) highlighted. (E) Proposed components of the ZSWIM8 cullin-RING ubiquitin ligase.

Fig. 2.. ZSWIM8 ubquitin ligase components are essential for TDMD.

(A) Flow cytometry analysis of EGFP expression in CYRANO^+/+ K562 EGFP^miR−7 cells after lentiviral expression of Cas9 and sgRNAs targeting the indicated genes. (B) Northern blot analysis of miRNA expression in CYRANO^+/+ and CYRANO^−/− K562 cells after expression of sgRNAs targeting ZSWIM8 CRL components. (C) Schematic of reprogrammed mCherry-NREP transcript (NREP_29a/b) and its predicted base-pairing with miR-29a (upper) and miR-29b (lower). (D) Predicted base-pairing of the mutant NREP transcript with seed binding only (NREP_seed) with miR-29a and miR-29b. (E) Northern blot analysis of miRNA expression in HCT116 cells expressing NREP transcripts and the indicated sgRNAs. n=3 biological replicates for northern blot and flow cytometry experiments (representative data shown).

Fig. 3.. The ZSWIM8 ubquitin ligase interacts with AGO2 and is required for proteasome-dependent miRNA turnover.

(A) Co-immunoprecipitation of V5-ZSWIM8 with CRL components and AGO2. (B) Western blot analysis of streptavidin pull-downs from cells expressing V5-TbID or V5-ZSWIM8-TbID. (C-D) Northern blot analysis of K562 cells of the indicated genotypes following 24 or 48 hours of treatment with lysosome inhibitor bafilomycin (200 nM) or proteasome inhibitor bortezomib (2 μM) (C) or NEDDylation inhibitor MLN4924 (5 μM) (D). n=3 biological replicates for all experiments (representative results shown).

Fig. 4.. Surface-exposed lysines in AGO2 are required for TDMD.

(A) Structure of human AGO2 engaged with a TDMD-inducing target (PDB: 6NIT) (16), with positions of mutated surface exposed lysines highlighted in cyan or red (K493). (B) Northern blot of the indicated miRNAs in HCT116 AGO1/2/3^−/− cells reconstituted with wild-type FLAG-HA-tagged AGO2 (FH-AGO2) or FH-AGO2^KR25. (C) qRT-PCR analysis of miR-7 levels (normalized to miR-16 levels) in AGO1/2/3^−/− cells reconstituted with the indicated FH-AGO2 constructs, with or without knockout of ZSWIM8. Mean +/− SD shown. **p<0.01; Student’s t test. (D) Northern blot of the indicated miRNAs in AGO1/2/3^−/− cells reconstituted with wild-type or mutant FH-AGO2. Mutants shown in this panel exhibited a statistically significant increase in miR-7 abundance in qRT-PCR experiments (see Fig. S6H). n=3 biological replicates for all experiments (representative northern blots shown).

Fig. 5.. miRNA tailing and trimming is not essential for TDMD.

Northern and western blot analyses of AGO2-loaded miRNAs (miR-7, miR-16) and AGO2, respectively, after transfection of the indicated miR-7 duplexes into miR-7–1^−/− K562 cells and immunoprecipitation of AGO2 at successive time-points. miR-7 levels were quantified and normalized to miR-16 (bottom panels). Results shown are representative of biological duplicate experiments.

Fig. 6.. The ZSWIM8 ubiquitin ligase complex regulates miRNA expression in diverse cell lines.

Small RNA sequencing of CYRANO^−/− K562, ZSWIM8^−/− K562, ZSWIM8^−/− HEK293T, or ZSWIM8^−/− MEFs, along with associated wild-type cells (n=3 biological replicates per genotype). miR-7–5p is the only significantly upregulated miRNA in CYRANO^−/− cells, whereas additional upregulated miRNAs were observed in ZSWIM8^−/− cells without an increase in the corresponding passenger strands. FC, fold change; CPM, counts per million.

Fig. 7.. Proposed model of ZSWIM8 ubquitin ligase activity in TDMD.

The schematic depicts the ZSWIM8 complex ubiquitylating AGO, and possibly other associated proteins, when they are engaged with a TDMD target, leading to proteasomal degradation of the miRNA-containing complex. Release of the miRNA may lead to its degradation by cytoplasmic RNases while the target transcript is recycled for another round of TDMD. B: Elongin B; C: Elongin C; E2: Ubiquitin-conjugating enzyme; Ub: Ubiquitin.