Life of RISC: Formation, action, and degradation of RNA-induced silencing complex.

Small RNAs regulate a wide variety of biological processes by repressing the expression of target genes at the transcriptional and post-transcriptional levels. To achieve these functions, small RNAs form RNA-induced silencing complex (RISC) together with a member of the Argonaute (AGO) protein family. RISC is directed by its bound small RNA to target complementary RNAs and represses their expression through mRNA cleavage, degradation, and/or translational repression. Many different factors fine-tune RISC activity and stability-from guide-target RNA complementarity to the recruitment of other protein partners to post-translational modifications of RISC itself. Here, we review recent progress in understanding RISC formation, action, and degradation, and discuss new, intriguing questions in the field.

Plant 22-nt siRNAs mediate translational repression and stress adaptation.

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation2,3, respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here we report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases-NIA1 and NIA2-produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2, which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, our research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses.

The clade-specific target recognition mechanisms of plant RISCs.

Eukaryotic Argonaut proteins (AGOs) assemble RNA-induced silencing complexes (RISCs) with guide RNAs that allow binding to complementary RNA sequences and subsequent silencing of target genes. The model plant Arabidopsis thaliana encodes 10 different AGOs, categorized into three distinct clades based on amino acid sequence similarity. While clade 1 and 2 RISCs are known for their roles in post-transcriptional gene silencing, and clade 3 RISCs are associated with transcriptional gene silencing in the nucleus, the specific mechanisms of how RISCs from each clade recognize their targets remain unclear. In this study, I conducted quantitative binding analyses between RISCs and target nucleic acids with mismatches at various positions, unveiling distinct target binding characteristics unique to each clade. Clade 1 and 2 RISCs require base pairing not only in the seed region but also in the 3' supplementary region for stable target RNA binding, with clade 1 exhibiting a higher stringency. Conversely, clade 3 RISCs tolerate dinucleotide mismatches beyond the seed region. Strikingly, they bind to DNA targets with an affinity equal to or surpassing that of RNA, like prokaryotic AGO complexes. These insights challenge existing views on plant RNA silencing and open avenues for exploring new functions of eukaryotic AGOs.

Functional specialization of monocot DCL3 and DCL5 proteins through the evolution of the PAZ domain.

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt small interfering RNAs (siRNAs), heterochromatic siRNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs), respectively. The former small RNAs are linked to silencing of transposable elements and heterochromatic repeats, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient 'eudicot-type' DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 5' phosphates and 3' overhangs, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5, which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation.

The model plant Arabidopsis thaliana encodes as many as ten Argonaute proteins (AGO1-10) with different functions. Each AGO selectively loads a set of small RNAs by recognizing their length and 5' nucleotide identity to properly regulate target genes. Previous studies showed that AGO4 and AGO6, key factors in DNA methylation, incorporate 24-nt small-interfering RNAs with 5' adenine (24A siRNAs). However, it has been unclear how these AGOs specifically load 24A siRNAs. Here, we biochemically investigated the siRNA preference of AGO4, AGO6 and their chimeric mutants. We found that AGO4 and AGO6 use distinct mechanisms to preferentially load 24A siRNAs. Moreover, we showed that the 5' A specificity of AGO4 and AGO6 is not determined by the previously known nucleotide specificity loop in the MID domain but rather by the coordination of the MID and PIWI domains. These findings advance our mechanistic understanding of how small RNAs are accurately sorted into different AGO proteins in plants.

Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants.

Secondary small interfering RNA (siRNA) production, triggered by primary small RNA targeting, is critical for proper development and antiviral defense in many organisms. RNA-dependent RNA polymerase (RDR) is a key factor in this pathway. However, how RDR specifically converts the targets of primary small RNAs into double-stranded RNA (dsRNA) intermediates remains unclear. Here, we develop an in vitro system that allows for dissection of the molecular mechanisms underlying the production of trans-acting siRNAs, a class of plant secondary siRNAs that play roles in organ development and stress responses. We find that a combination of the dsRNA-binding protein, SUPPRESSOR OF GENE SILENCING3; the putative nuclear RNA export factor, SILENCING DEFECTIVE5, primary small RNA, and Argonaute is required for physical recruitment of RDR6 to target RNAs. dsRNA synthesis by RDR6 is greatly enhanced by the removal of the poly(A) tail, which can be achieved by the cleavage at a second small RNA-binding site bearing appropriate mismatches. Importantly, when the complementarity of the base pairing at the second target site is too strong, the small RNA-Argonaute complex remains at the cleavage site, thereby blocking the initiation of dsRNA synthesis by RDR6. Our data highlight the light and dark sides of double small RNA targeting in the secondary siRNA biogenesis.

MicroRNAs block assembly of eIF4F translation initiation complex in Drosophila.

miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. We have previously shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. Here, we examined the assembly of translation initiation factors on the target mRNA under repression. We show that Ago1-RISC induces dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. We propose that miRNAs act to block the assembly of the eIF4F complex during translation initiation.

Molecular insights into microRNA-mediated translational repression in plants.

microRNAs (miRNAs) bind Argonaute proteins in order to form RNA-induced silencing complexes (RISCs) that can silence the expression of complementary mRNAs. Plant miRNAs can mediate the cleavage of their target mRNAs as well as the repression of their translation. Here, by using an in vitro system prepared from plant culture cells, we biochemically dissect the mechanisms by which Arabidopsis thaliana ARGONAUTE1 RISC (AtAGO1-RISC) silences its mRNA targets. We find that AtAGO1-RISC has the ability to repress translation initiation without promoting deadenylation or mRNA decay. Strikingly, AtAGO1-RISC bound in the 5' untranslated region or the open reading frame can sterically block the recruitment or movement of ribosomes. These silencing effects require more extensive base pairing to the target site in comparison to typical animal miRNAs. Our data provide mechanistic insights into miRNA-mediated translational repression in plants.

Biochemical and single-molecule analyses of the RNA silencing suppressing activity of CrPV-1A.

Viruses often encode viral silencing suppressors (VSSs) to counteract the hosts' RNA silencing activity. The cricket paralysis virus 1A protein (CrPV-1A) is a unique VSS that binds to a specific Argonaute protein (Ago)-the core of the RNA-induced silencing complex (RISC)-in insects to suppress its target cleavage reaction. However, the precise molecular mechanism of CrPV-1A action remains unclear. Here we utilized biochemical and single-molecule imaging approaches to analyze the effect of CrPV-1A during target recognition and cleavage by Drosophila Ago2-RISC. Our results suggest that CrPV-1A obstructs the initial target searching by Ago2-RISC via base pairing in the seed region. The combination of biochemistry and single-molecule imaging may help to pave the way for mechanistic understanding of VSSs with diverse functions.

Arabidopsis ARGONAUTE7 selects miR390 through multiple checkpoints during RISC assembly.

Plant ARGONAUTE7 (AGO7) assembles RNA-induced silencing complex (RISC) specifically with miR390 and regulates the auxin-signalling pathway via production of TAS3 trans-acting siRNAs (tasiRNAs). However, how AGO7 discerns miR390 among other miRNAs remains unclear. Here, we show that the 5' adenosine of miR390 and the central region of miR390/miR390* duplex are critical for the specific interaction with AGO7. Furthermore, despite the existence of mismatches in the seed and central regions of the duplex, cleavage of the miR390* strand is required for maturation of AGO7-RISC. These findings suggest that AGO7 uses multiple checkpoints to select miR390, thereby circumventing promiscuous tasiRNA production.

Poly(A)-binding protein facilitates translation of an uncapped/nonpolyadenylated viral RNA by binding to the 3' untranslated region.

Viruses employ an alternative translation mechanism to exploit cellular resources at the expense of host mRNAs and to allow preferential translation. Plant RNA viruses often lack both a 5' cap and a 3' poly(A) tail in their genomic RNAs. Instead, cap-independent translation enhancer elements (CITEs) located in the 3' untranslated region (UTR) mediate their translation. Although eukaryotic translation initiation factors (eIFs) or ribosomes have been shown to bind to the 3'CITEs, our knowledge is still limited for the mechanism, especially for cellular factors. Here, we searched for cellular factors that stimulate the 3'CITE-mediated translation of Red clover necrotic mosaic virus (RCNMV) RNA1 using RNA aptamer-based one-step affinity chromatography, followed by mass spectrometry analysis. We identified the poly(A)-binding protein (PABP) as one of the key players in the 3'CITE-mediated translation of RCNMV RNA1. We found that PABP binds to an A-rich sequence (ARS) in the viral 3' UTR. The ARS is conserved among dianthoviruses. Mutagenesis and a tethering assay revealed that the PABP-ARS interaction stimulates 3'CITE-mediated translation of RCNMV RNA1. We also found that both the ARS and 3'CITE are important for the recruitment of the plant eIF4F and eIFiso4F factors to the 3' UTR and of the 40S ribosomal subunit to the viral mRNA. Our results suggest that dianthoviruses have evolved the ARS and 3'CITE as substitutes for the 3' poly(A) tail and the 5' cap of eukaryotic mRNAs for the efficient recruitment of eIFs, PABP, and ribosomes to the uncapped/nonpolyadenylated viral mRNA.

Mechanisms that regulate the production of secondary siRNAs in plants.

Many organisms produce secondary small interfering RNAs (siRNAs) that are triggered by primary small RNAs to regulate various biological processes. Plants have evolved several types of secondary siRNA biogenesis pathways that play important roles in development, stress responses and defense against viruses and transposons. The critical step of these pathways is the production of double-stranded RNAs by RNA-dependent RNA polymerases. This step is normally tightly regulated, but when its control is released, secondary siRNA production is initiated. In this article, we will review the recent advances in secondary siRNA production triggered by microRNAs encoded in the genome and siRNAs derived from invasive nucleic acids. In particular, we will focus on the factors, events, and RNA/DNA elements that promote or inhibit the early steps of secondary siRNA biogenesis.

Template recognition mechanisms by replicase proteins differ between bipartite positive-strand genomic RNAs of a plant virus.

Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3' untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.

Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants.

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

A viral noncoding RNA generated by cis-element-mediated protection against 5'->3' RNA decay represses both cap-independent and cap-dependent translation.

Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3' untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3' UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5'-->3' decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3'-side RNA sequences against 5'-->3' decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.

[Viral noncoding RNAs].

Many lines of recent evidence indicate that non-coding RNAs including micro RNAs (miRNAs) and small interfering RNAs (siRNAs) play an important role in the control of gene expression in diverse cellular processes and in defense responses against molecular parasites such as viruses and transposons. Viruses also use many different types of non-coding RNAs for regulating expression of their own genome or host genome temporally and spatially to ensure efficient virus proliferation and/or latency in the host cell. Here, we introduce the generation mechanisms and functions of novel non-coding RNAs generated from both animal and plant RNA viruses, after a brief review of non-coding RNAs of DNA viruses.

In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6.

RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.

In Vitro Analysis of ARGONAUTE-Mediated Target Cleavage and Translational Repression in Plants.

MicroRNAs (miRNAs) are endogenous small RNAs, which negatively regulate expression of complementary target genes at the post-transcriptional level. In plants, miRNAs are mainly loaded onto ARGONAUTE1 to form RNA-induced silencing complexes (RISCs), which mediate target mRNA cleavage as well as translational repression. The cell-free system derived from tobacco BY-2 protoplasts has become a powerful tool not only for the analysis of RISC assembly mechanism but also for mechanistic dissection of plant RISC functions. Here we describe the detailed protocols for the preparation of BY-2 cell lysate and the procedure to analyze the dual function of plant RISC-target cleavage and translational repression-in vitro.

The poly(A) tail blocks RDR6 from converting self mRNAs into substrates for gene silencing.

It remains unclear how post-transcriptional gene silencing (PTGS) in plants discriminates aberrant RNAs from canonical messenger RNAs (mRNAs). The key step of plant PTGS is the conversion of aberrant RNAs into double-stranded RNAs by RNA-DEPENDENT RNA POLYMERASE6 (RDR6). Here, we show that RDR6 itself selects aberrant poly(A)-less mRNAs over canonical polyadenylated mRNAs as templates at the initiation step of complementary strand synthesis. This mechanism can be viewed as an innate safeguard against 'self-attack' by PTGS.

Requirement for eukaryotic translation initiation factors in cap-independent translation differs between bipartite genomic RNAs of red clover necrotic mosaic virus.

The bipartite genomic RNAs of red clover necrotic mosaic virus (RCNMV) lack a 5' cap and a 3' poly(A) tail. RNA1 encodes viral replication proteins, and RNA2 encodes a movement protein (MP). These proteins are translated in a cap-independent manner. We previously identified two cis-acting RNA elements that cooperatively recruit eukaryotic translation initiation factor (eIF) complex eIF4F or eIFiso4F to RNA1. Such cis-acting RNA elements and host factors have not been identified in RNA2. Here we found that translation of RNA1 was significantly compromised in Arabidopsis thaliana carrying eif4f mutation. RNA1 replicated efficiently in eifiso4f1 mutants, suggesting vigorous translation of the replication proteins from RNA1 in the plants. In contrast, MP accumulation was decreased in eifiso4f1 mutants but not in eif4f mutants. Collectively, these results suggest that RCNMV uses different eIF complexes for translation of its bipartite genomic RNAs, which may contribute to fine-tuning viral gene expression during infection.