Graphical Abstract Highlights d Empty but not miRNA-loaded fly Ago1 undergoes selective ubiquitination d The RING-type E3 ubiquitin ligase Iruka preferentially recognizes empty Ago1 d Iruka ubiquitinates Lys514 of Ago1, which is only accessible in the empty state d Iruka eliminates dysfunctional Ago1 to ensure effective miRNA-mediated silencing SUMMARYMicroRNAs (miRNAs) are loaded into the Argonaute subfamily of proteins (AGO) to form an effector complex that silences target genes. Empty but not miRNA-loaded AGO is selectively degraded across species. However, the mechanism and biological significance of selective AGO degradation remain unclear. We discovered a RING-type E3 ubiquitin ligase we named Iruka (Iru), which selectively ubiquitinates the empty form of Drosophila Ago1 to trigger its degradation. Iru preferentially binds empty Ago1 and ubiquitinates Lys514 in the L2 linker, which is predicted to be inaccessible in the miRNA-loaded state. Depletion of Iru results in global impairment of miRNA-mediated silencing of target genes and in the accumulation of aberrant Ago1 that is dysfunctional for canonical protein-protein interactions and miRNA loading. Our findings reveal a sophisticated mechanism for the selective degradation of empty AGO that underlies a quality control process to ensure AGO function. R (3.4.0) N/A https://www.r-project.org/ TargetScan (Fly, Release 6.2) Ruby et al., 2007 http://www.targetscan.org/fly_12/ Bowtie Langmead et al.

Highlights d The dsRNA binding protein SGS3 is required for microRNAmediated ribosome stalling d SGS3 interacts directly with the 3 0 end of the AGO-loaded microRNA on the target d SGS3-microRNA-AGO can pause ribosomes on cleavable and non-cleavable sites d Ribosome pausing correlates positively with production of secondary siRNAs

Highlights d The empty state of Drosophila Ago1 is degraded by autophagy d Empty Ago1 is recognized by VCP, a mediator for selective autophagy d The Ufd1-Npl4 heterodimer links empty Ago1 and VCP d VCP-Ufd1-Npl4 machinery ensures proper gene silencing by microRNAs

The path of ribosomes on mRNAs can be impeded by various obstacles. One such example is halting of ribosome movement by microRNAs, though the exact mechanism and physiological role remain unclear. Here, we find that ribosome stalling caused by the Argonaute-miRNA-SGS3 complex regulates production of secondary siRNA biogenesis in plants. We show that the double-stranded RNA-binding protein, SGS3, directly interacts with the 3′ end of the microRNA-Argonaute complex, resulting in ribosome stalling. Strikingly, microRNA-mediated ribosome stalling enhances production of secondary small interfering RNAs (siRNAs) from target mRNAs. Our results uncover a previously uncharacterized role for paused ribosomes in regulation of small RNA function that may have broad biological implications across the plant kingdom.