Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs

ARGONAUTE (AGO) RNA-binding proteins are involved in RNA silencing. They bind to short interfering RNAs (siRNAs) and microRNAs (miRNAs) through a conserved PAZ domain, and, in animals, they assemble into a multisubunit RNA-induced silencing complex (RISC). The mammalian AGO2, termed Slicer, directs siRNA- and miRNA-mediated cleavage of a target RNA. In Arabidopsis, there are 10 members of the AGO family, and the AGO1 protein is potentially the Slicer component in different RNA-silencing pathways. Here, we show that AGO1 selectively recruits certain classes of short silencing-related RNA. AGO1 is physically associated with miRNAs, transacting siRNAs, and transgene-derived siRNAs but excludes virus-derived siRNAs and 24-nt siRNAs involved in chromatin silencing. We also show that AGO1 has Slicer activity. It mediates the in vitro cleavage of a mir165 target RNA in a manner that depends on the sequence identity of amino acid residues in the PIWI domain that are predicted by homology with animal Slicer-competent AGO proteins to constitute the RNase catalytic center. However, unlike animals, we find no evidence that AGO1 Slicer is in a high molecular weight RNA-induced silencing complex. The Slicer activity fractionates as a complex of approximately 150 kDa that likely constitutes the AGO1 protein and associated RNA without any other proteins. Based on sequence similarity, we predict that other Arabidopsis AGOs might have a similar catalytic activity but recruit different subsets of siRNAs or miRNAs.     

MicroRNA在疾病诊断和治疗中的研究进展

MicroRNA (miRNA)是近年发现的一类大小约21～22个核苷酸的非编码小RNA分子,它主要通过RNA诱导的沉默复合体(RNA-induced silencing complex,RISC)来调控基因的表达,参与调控机体的各种生理功能.因此,miRNA的功能失调与人类疾病的发生、发展密切相关.该文就miRNA在多种疾病的诊断和治疗方面的研究进展作一综述.     

From the Cover: 22-nucleotide RNAs trigger secondary siRNA biogenesis in plants

The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediated transient expression in Nicotiana benthamiana confirms that the siRNA-initiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.     

Isoprenoid biosynthesis is required for miRNA function and affects membrane association of ARGONAUTE 1 in Arabidopsis

Plant and metazoan microRNAs (miRNAs) guide ARGONAUTE (AGO) protein complexes to regulate expression of complementary RNAs via base pairing. In the plant Arabidopsis thaliana, the main miRNA effector is AGO1, but few other factors required for miRNA activity are known. Here, we isolate the genes defined by the previously described miRNA action deficient (mad) mutants, mad3 and mad4. Both genes encode enzymes involved in isoprenoid biosynthesis. MAD3 encodes 3-hydroxy-3-methylglutaryl CoA reductase (HMG1), which functions in the initial C(5) building block biogenesis that precedes isoprenoid metabolism. HMG1 is a key regulatory enzyme that controls the amounts of isoprenoid end products. MAD4 encodes sterol C-8 isomerase (HYDRA1) that acts downstream in dedicated sterol biosynthesis. Using yeast complementation assays and in planta application of lovastatin, a competitive inhibitor of HMG1, we show that defects in HMG1 catalytic activity are sufficient to inhibit miRNA activity. Many isoprenoid derivatives are indispensable structural and signaling components, and especially sterols are essential membrane constituents. Accordingly, we provide evidence that AGO1 is a peripheral membrane protein. Moreover, specific hypomorphic mutant alleles of AGO1 display compromised membrane association and AGO1-membrane interaction is reduced upon knockdown of HMG1/MAD3. These results suggest a possible basis for the requirement of isoprenoid biosynthesis for the activity of plant miRNAs, and unravel mechanistic features shared with their metazoan counterparts.     

In vitro reconstitution of the human RISC-loading complex

Targeted gene silencing by RNAi requires the RNA-induced silencing complex (RISC), whose core component is the protein Argonaute (Ago) bound to a microRNA (miRNA) or an siRNA. In humans, Ago2 is loaded with miRNAs by the action of a specialized assembly called the RISC-loading complex (RLC), comprising the proteins Ago2, Dicer, and TRBP. Here we show that the human RLC assembles spontaneously in vitro from purified components. No cofactors or chaperones are required for the complex to form. The reconstituted RLC, containing one copy of each protein, has the dicing, slicing, guide-strand selection, and Ago2-loading activities observed for the endogenous RLC. Furthermore, once Ago2 is loaded with an miRNA, it tends to dissociate from the rest of the complex. These results lay the groundwork for future structural and functional dissection of RISC loading in humans.     

RNA, but not protein partners, is directly responsible for translational silencing by a bacterial Hfq-binding small RNA

SgrS is an Hfq-binding small RNA that is induced under glucose phosphate stress in Escherichia coli. It forms a specific ribo nucleo protein complex with Hfq and RNase E resulting in translational repression and rapid degradation of ptsG mRNA, encoding the glucose transporter. Here, we report translational silencing of ptsG mRNA in a defined in vitro system. We demonstrate that SgrS and Hfq are the minimum components for translational silencing to faithfully reproduce the reaction in cells. We show that ptsG-SgrS base pairing is sufficient to cause translational repression when the ptsG mRNA is forced to base pair with SgrS without the help of Hfq. The extent of translational repression correlates with the extent of duplex formation. We conclude that base pairing itself but not Hfq is directly responsible for translational silencing and the major role of Hfq in gene silencing is to stimulate the base pairing between SgrS and ptsG mRNA. This simple mechanism is in striking contrast to miRNA action in eukaryote in which the RNA is believed to act only as a guide of protein partners.     

RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex

Tomato bushy stunt virus (TBSV) and other tombusviruses encode a p19 protein (P19), which is a suppressor of RNAi. Wild-type TBSV or p19-defective mutants initially show a similar infection course in Nicotiana benthamiana, but the absence of an active P19 results in viral RNA degradation followed by recovery from infection. P19 homodimers sequester 21-nt virus-derived duplex siRNAs, and it is thought that this prevents the programming of an antiviral RNA-induced silencing complex to avoid viral RNA degradation. Here we report on chromatographic fractionation (gel filtration, ion exchange, and hydroxyapatite) of extracts from healthy or infected Nicotiana benthamiana plants in combination with in vitro assays for ribonuclease activity and detection of TBSV-derived siRNAs. Only extracts of plants infected with p19 mutants provided a source of sequence-nonspecific but ssRNA-targeted in vitro ribonuclease activity that coeluted with components of a wide molecular weight range. In addition, we isolated a discrete approximately 500-kDa protein complex that contained approximately 21-nt TBSV-derived siRNAs and that exhibited ribonuclease activity that was TBSV sequence-preferential, ssRNA-specific, divalent cation-dependent, and insensitive to a ribonuclease inhibitor. We believe that this study provides biochemical evidence for a virus-host system that infection in the absence of a fully active RNAi suppressor induces ssRNA-specific ribonuclease activity, including that conferred by a RNA-induced silencing complex, which is likely the cause for the recovery of plants from infection.     

TRIM65 regulates microRNA activity by ubiquitination of TNRC6

MicroRNAs (miRNAs) are small evolutionarily conserved regulatory RNAs that modulate mRNA stability and translation in a wide range of cell types. MiRNAs are involved in a broad array of biological processes, including cellular proliferation, differentiation, and apoptosis. To identify previously unidentified regulators of miRNA, we initiated a systematic discovery-type proteomic analysis of the miRNA pathway interactome in human cells. Six of 66 genes identified in our proteomic screen were capable of regulating lethal-7a (let-7a) miRNA reporter activity. Tripartite motif 65 (TRIM65) was identified as a repressor of miRNA activity. Detailed analysis indicates that TRIM65 interacts and colocalizes with trinucleotide repeat containing six (TNRC6) proteins in processing body-like structures. Ubiquitination assays demonstrate that TRIM65 is an ubiquitin E3 ligase for TNRC6 proteins. The combination of overexpression and knockdown studies establishes that TRIM65 relieves miRNA-driven suppression of mRNA expression through ubiquitination and subsequent degradation of TNRC6.MicroRNAs (miRNAs) are small noncoding RNAs that regulate the translation and stability of mRNA in animals and plants (1, 2). The canonical biogenesis of miRNAs starts with a hairpin-like primary miRNA (primiRNA), typically a product of RNA polymerase II (3, 4). In the nucleus, the DROSHA/DGCR8 microprocessor complex recognizes and cleaves the primiRNA hairpin, which leads to release of a precursor miRNA (premiRNA) hairpin that is ∼55–70 nt in length (5–7). The premiRNA is exported to the cytoplasm by a complex of Exportin 5 and RAN-GTP (8, 9). In the cytoplasm, the premiRNA terminal loop is cleaved by DICER in collaboration with TARBP2, yielding ∼22-nt RNA duplexes. One strand of the duplex is preferentially incorporated into the RNA-induced silencing complex (RISC), where the miRNA and mRNA interact (3). In the RISC, miRNA targets mRNA for translational repression, deadenylation, or degradation (10–12).The RISC minimally consists of two core protein components, Argonaute (AGO) and trinucleotide repeat containing six (TNRC6; also known as GW182) proteins or their paralogs, which are key factors for function of the RISC. These proteins localize in specialized cytoplasmic foci known as mRNA processing bodies (P-bodies) (13, 14). P-bodies also contain effector molecules that facilitate mRNA degradation, including decapping enzymes (DCP1 and DCP2) required for miRNA-mediated gene silencing (15) and the CCR4–NOT deadenylase complex, which removes poly(A) from messages destabilized by miRNA (15).The core proteins that participate in miRNA biogenesis and regulation have been identified (6, 16–20), but the global organization and coordination of this system are incompletely understood. To explore the miRNA protein–protein interaction network systematically, we initiated a global proteomic analysis of the miRNA pathway interactome (MPI) in human cells. Analysis of 40 MPI-associated baits revealed connections with 363 proteins, forming a framework of 499 unique protein interactions. RNAi screening of 66 previously unidentified proteins associated with the MPI identified tripartite motif 65 (TRIM65) and five other regulators of miRNA activity. TRIM65 is an ubiquitin E3 ligase that colocalizes with TNRC6 paralogs and triggers proteasome-dependent degradation of TNRC6 proteins. TRIM65 represents a previously unidentified member of the miRNA pathway, which negatively regulates the miRNA-guided mRNA silencing machinery.     

Comprehensive analysis of mammalian miRNA* species and their role in myeloid cells

Processing of pre-miRNA through Dicer1 generates an miRNA duplex that consists of an miRNA and miRNA* strand. Despite the general view that miRNA*s have no functional role, we further investigated miRNA* species in 10 deep-sequencing libraries from mouse and human tissue. Comparisons of miRNA/miRNA* ratios across the miRNA sequence libraries revealed that 50% of the investigated miRNA duplexes exhibited a highly dominant strand. Conversely, 10% of miRNA duplexes showed a comparable expression of both strands, whereas the remaining 40% exhibited variable ratios across the examined libraries, as exemplified by miR-223/miR-223* in murine and human cell lines. Functional analyses revealed a regulatory role for miR-223* in myeloid progenitor cells, which implies an active role for both arms of the miR-223 duplex. This was further underscored by the demonstration that miR-223 and miR-223* targeted the insulin-like growth factor 1 receptor/phosphatidylinositol 3-kinase axis and that high miR-223* levels were associated with increased overall survival in patients with acute myeloid leukemia. Thus, we found a supporting role for miR-223* in differentiating myeloid cells in normal and leukemic cell states. The fact that the miR-223 duplex acts through both arms extends the complexity of miRNA-directed gene regulation of this myeloid key miRNA.