Small RNA sorting: matchmaking for Argonautes

Abstract

Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes - microRNAs (miRNAs) and small interfering RNAs (siRNAs) - in plants and animals.

Figure 1. MicroRNA biogenesis in Drosophila melanogaster

MicroRNAs (miRNAs) are generally transcribed by RNA polymerase II (RNAPII) to yield primary miRNAs (pri-miRNAs). pri-miRNAs are cropped in the nucleus by Drosha–Pasha complexes to release shorter precursor miRNAs (pre-miRNAs). miRNAs that reside within short introns of protein-coding genes are excised by the splicing machinery and are termed mirtrons. Following linearization of mirtron intermediates by the lariat-debranching enzyme, they fold into pre-miRNAs. Some 3′-tailed mirtrons undergo further trimming by the exosome. pre-miRNAs are transported to the cytoplasm by Exportin 5 (EXP5), where further processing takes place. Dicer 1 (DCR1), in collaboration with an isoform of its dsRNA binding domain protein partner Loquacious (LOQS-PB), liberates miR:miR* duplexes that dissociate from DCR1 for downstream sorting.

Figure 2. Production of small interfering RNAs

a | In flies, perfect or nearly perfect dsRNA precursors of varying origin and structure are processed in the cytoplasm by the RNase III enzyme Dicer 2 (DCR2) and its co-factor, an isoform of Loquacious (LOQS-PD), to yield small interfering RNA (siRNA) duplexes that contain guide and passenger strands. b | Caenorhabditis elegans primary siRNAs are processed from long dsRNA triggers through the action of DCR-1. These primary siRNAs associate with the Argonaute family protein, RDE-1, and guide it to target transcripts. The RDE-1–target interaction recruits an RNA-dependent RNA polymerase (RdRP), which uses the target as template for the de novo synthesis of secondary siRNAs that feature 5′ triphosphate ends (see main text for further details). c | The production of Arabidopsis thaliana trans-acting siRNAs (ta-siRNAs) requires the interplay of canonical components of the microRNA (miRNA) and siRNA biogenesis machineries. The process is triggered by miRNA-mediated cleavage of non-coding TAS transcripts by miR390–AGO7 or miR173–AGO1, respectively. Slicing triggers the recruitment of SUPPRESSOR OF GENE SILENCING (SGS3) and RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), which synthesize dsRNA using the cleavage site as the entry point. The resulting dsRNA is processed by DICER-LIKE 4 (DCL4) and its dsRBD protein partner DRB4 into a phased series of 21-nucleotide (nt) siRNA duplexes. ta-siRNAs are methylated by HUA ENHANCER 1 (HEN1) before AGO loading.

Figure 3. Small RNA sorting and RNA-induced silencing complex assembly in flies

a | Structural determinants dominate the decision to sort small RNAs into fly Argonaute 1 (AGO1) or AGO2. AGO1-biased (usually microRNA (miRNA)) duplexes contain several bulges and mismatches, especially in the central region of the duplex. Mature miR strands show a strong bias for a terminal U. By contrast, AGO2-biased (usually siRNA) duplexes show extensive base pairing. Loaded guide strands often start with C. b | Unloaded AGO1 is recognized and bound by the heat shock cognate 70 (HSC70)–heat shock protein 90 (HSP90) chaperone complex and, following binding of ATP, adopts an ‘open’ conformational state. Loading-competent AGO1 receives miRNA duplexes containing several mismatches. The incorporation of duplexes into AGO1 is likely aided by as yet unidentified loading factors. ATP hydrolysis results in dissociation of the chaperone complex from AGO1, followed by passive unwinding of the duplex, a process promoted by mismatches. The miR* strand is degraded following unwinding. c | The HSC70–HSP90 chaperone complex associates with unloaded AGO2. Binding of ATP to the chaperone complex leads to conformational changes that allow AGO2 to receive small duplexes from the AGO2-loading machinery. Small RNA duplexes with perfect or near-perfect base-pairing (especially those with good pairing in the central region) are recognized by Dicer 2 (DCR2) and its co-factor R2D2 (AGO2–RISC-loading machinery) and inserted into AGO2. The chaperone complex dissociates following ATP hydrolysis, causing a change in the conformation of AGO2. Following passenger strand slicing by AGO2, component 3 promoter of RISC (C3PO) degrades the cleavage products. Subsequently, the 3′ terminus of the guide strand is methylated by HUA ENHANCER 1 (HEN1) to yield mature AGO2–RISC. SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.