Origins and Mechanisms of miRNAs and siRNAs

Abstract

Over the last decade, approximately 20-30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. Two primary categories of these small RNAs--short interfering RNAs (siRNAs) and microRNAs (miRNAs)--act in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment.

Figure 1. Core Features of miRNA and siRNA Silencing

(A) Common aspects of all miRNA and siRNA pathways. Double-stranded RNA precursors of various kinds are processed by a Dicer protein into short (∼20−30 nt) fragments. One strand of the processed duplex is loaded into an Argonaute protein, enabling target RNA recognition through Watson-Crick base pairing. Once the target is recognized, its expression is modulated by one of several distinct mechanisms, depending on the biological context. (B) Dicer proteins cleave dsRNA precursors into characteristic lengths through the action of two RNase III domains. The domain arrangement of most Dicer enzymes is shown at the top. Processing occurs most readily at dsRNA ends, which associate with the PAZ domain present in most Dicer enzymes. The substrate is then positioned within the active sites of the RNase III domains, which cleave the ∼20−30 nt miRNA/siRNA duplex from its precursor. This model is supported by the crystal structure of Giardia Dicer, shown with a dsRNA modeled into the structure (image kindly provided by J. Doudna). In addition to the canonical PAZ and RNase III domains, the structure shows active-site metal ions (purple) and a “ruler” helix (red) that helps to specify the length of the siRNA product. (C) Argonaute proteins are RNA silencing effectors that are guided to their targets by short single-stranded nucleic acids. The canonical arrangement of Ago domains is given at the top. Below is a crystal structure of the Thermus thermophilus Ago protein, with a bound DNA guide strand base paired to an RNA target. The 5′ end of the guide strand associates with a binding pocket in the Mid domain, and the 3′ end binds the PAZ domain. The target cleavage site is juxtaposed with active-site residues in the PIWI domain, though in this case cleavage is suppressed by mismatches between the guide and the target. (Structure reprinted with permission from Macmillan Publishers Ltd.: Wang et al. [2008]. Nature 456, 921−926.)

Figure 2. A Diversity of siRNA Sources

Several different categories of transcripts can adopt dsRNA structures that can be processed by Dicer into siRNAs. These duplexes can be intraor intermolecular, and although most are perfectly base paired, some (e.g., hairpin RNAs and gene/pseudogene duplexes) are not. An siRNA consists of a guide strand (red), which assembles into functional siRISC, and a passenger strand (blue), which is ejected and degraded. All forms of siRISC contain the siRNA bound to an Ago protein, and many if not most forms of siRISC contain additional factors. Target RNAs are then recognized by base pairing, and silencing ensues through one of several mechanisms. In many species, the siRNA populations that engage a target can be amplified by the action of RNA-dependent RNA polymerase (RdRP) enzymes, strengthening and perpetuating the silencing response.

Figure 3. Mechanisms of siRNA Silencing

During canonical RNAi (lower right), siRISC recognizes a perfectly complementary mRNA, leading to Ago-catalyzed mRNA cleavage at a single site within the duplex. After cleavage, functional siRISC is regenerated, whereas the mRNA fragments are further degraded. siRNAs are also capable of recognizing targets with imperfect complementarity (upper right). In some cases, they can silence targets by miRNA-like mechanisms involving translational repression and exonucleolytic degradation, though the frequency with which natural siRNAs use these pathways is not clear. Finally, siRISC can direct heterochromatin formation (left) by associating with nascent transcripts and RNA polymerases (RNA Pol II in S. pombe and RNA Pol IV/V in A. thaliana). In plants, target engagement leads to the association or activation of a DNA methyltransferase (DMT) that methylates the DNA (lower left), leading to heterochromatin formation. In S. pombe and probably in animals (upper left), the pathway involves a histone methyltransferase (HMT) that methylates Lys9 of histone H3 (data not shown), thereby inducing heterochromatinization. In most eukaryotes other than insects and mammals, target recognition by siRISC induces the synthesis of secondary dsRNAs and siRNAs by RdRP enzymes (lower middle). The secondary dsRNAs are processed by Dicer into siRNAs, which add to the pool of siRISC. In nematodes, many of the secondary siRNAs arise as single-stranded, unprimed transcripts with 5′-triphosphates and do not require Dicer processing.

Figure 4. Biogenesis of miRNAs and Assembly into miRISC in Plants and Animals

Nuclear transcription leads to capped and polyadenylated pri-miRNAs. In plants, Dcl1 processes the RNA in succession. The order of processing is not certain. The terminal loop may first be excised or it might be the flanking segments that are cleaved first. The second processing step by Dcl1 yields a mature miRNA/miRNA* duplex that becomes methylated and exported from the nucleus. In animals, the pri-miRNA is processed by Drosha with the aid of DGCR8 to generate a pre-miRNA species. This is exported from the nucleus and processed by Dicer to form the mature miRNA/miRNA* duplex. After processing, miRNAs are assembled into miRISC. Only one strand of the duplex is stably associated with an miRISC complex—the miRNA strand is usually more strongly favored than the miRISC* strand, although there are exceptions.

Figure 5. Possible Mechanisms of miRISC-Mediated Repression

Nonrepressed mRNAs recruit initiation factors and ribosomal subunits and form circularized structures that enhance translation (top). When miRISCs bind to mRNAs, they can repress initiation at the cap recognition stage (upper left) or the 60S recruitment stage (lower left). Alternatively, they can induce deadenylation of the mRNA and thereby inhibit circularization of the mRNA (bottom). They can also repress a postinitiation stage of translation by inducing ribosomes to drop off prematurely (lower right). Finally, they can promote mRNA degradation by inducing deadenylation followed by decapping.