A structural perspective of the protein–RNA interactions involved in virus-induced RNA silencing and its suppression

Small RNAs, including small interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi-associated interfering RNAs (piRNAs), are powerful gene expression regulators. This RNA-mediated regulation results in sequence-specific inhibition of gene expression by translational repression and/or mRNA degradation. siRNAs and miRNAs are generated by RNase III enzymes and subsequently loaded into Argonaute protein, a key component of the RNA induced silencing complex (RISC), to form the core of the RNA silencing machinery. RNA silencing acts as an ancient cell defense system against molecular parasites, such as transgenes, viruses and transposons. RNA silencing also plays an important role in the control of development. In plants, RNA silencing serves as a potent antiviral defense system. In response, many viruses have developed strategies to suppress RNA silencing. The striking sequence diversity among viral suppressors suggests that different viral suppressors could target different components of the RNA silencing machinery at different steps in different suppressing modes. Significant progresses have been made in this field for the past 5 years on the basis of structural information derived from RNase III family proteins, Dicer fragments and homologs, Argonaute homologs and viral suppressors. In this paper, we will review the current progress on the understanding of molecular mechanisms of RNA silencing; highlight the structural principles determining the protein–RNA recognition events along the RNA silencing pathways and the suppression mechanisms displayed by viral suppressors.     

Argonaute protein identity and pairing geometry determine cooperativity in mammalian RNA silencing

Small RNAs loaded into Argonaute proteins direct silencing of complementary target mRNAs. It has been proposed that multiple, imperfectly complementary small interfering RNAs or microRNAs, when bound to the 3' untranslated region of a target mRNA, function cooperatively to silence target expression. We report that, in cultured human HeLa cells and mouse embryonic fibroblasts, Argonaute1 (Ago1), Ago3, and Ago4 act cooperatively to silence both perfectly and partially complementary target RNAs bearing multiple small RNA-binding sites. Our data suggest that for Ago1, Ago3, and Ago4, multiple, adjacent small RNA-binding sites facilitate cooperative interactions that stabilize Argonaute binding. In contrast, small RNAs bound to Ago2 and pairing perfectly to an mRNA target act independently to silence expression. Noncooperative silencing by Ago2 does not require the endoribonuclease activity of the protein: A mutant Ago2 that cannot cleave its mRNA target also silences noncooperatively. We propose that Ago2 binds its targets by a mechanism fundamentally distinct from that used by the three other mammalian Argonaute proteins.     

MicroRNA-binding is required for recruitment of human Argonaute 2 to stress granules and P-bodies

Argonaute proteins are the core components of the RNA-induced silencing complex, the central effector of the mammalian RNA interference pathway. In the cytoplasm, they associate with at least two types of cytoplasmic RNA granules; processing bodies and stress granules, which function in mRNA degradation and translational repression, respectively. The significance of Argonaute association with these RNA granules is not entirely clear but it is likely related to their activities within the RNAi pathway. Understanding what regulates targeting of Argonautes to RNA granules may provide clues as to their functions at these organelles. To this end, there are a number of conflicting reports that describe the role of small RNAs in targeting Argonaute proteins in mammalian cells. We employed quantitative microscopic analyses of human Argonaute 2 (hAgo2) mutants to study factors that govern localization of this RNA-binding protein to cytoplasmic RNA granules. We report, for the first time, that hAgo2 is recruited to stress granules as a consequence of its interaction with miRNAs. Moreover, loading of small RNAs onto hAgo2 is not required for its stability, suggesting that a pool of unloaded hAgo2 may exist for extended periods of time in the cytoplasm.     

小RNA与蛋白质的相互作用

小分子调控RNA,包括siRNA（small interfering RNA）、miRNA（microRNA）和piRNA（piwiinteracting RNA）、hsRNA（heterochromatin associatedsmall RNA）等,是当前生命科学研究的前沿热点。越来越多的证据表明,这些小分子RNA存在于几乎所有较高等的真核生物细胞中,对生物体具有非常重要的调控功能。它们通过各种序列特异性的RNA基因沉默作用,包括RNA干扰（RNAi）、翻译抑制、异染色质形成等,调控诸如生长发育、应激反应、沉默转座子等各种各样的细胞进程。随着对这些小分子调控RNA的发现,一些RNascⅢ酶家族成员、Argonaute蛋白质家族成员及RNA结合蛋白质等先后被鉴定为小RNA的胞内蛋白质合作者,参与小RNA的加工成熟和在细胞内行使功能。本综述简介一些RNA沉默作用途径中重要组分的结构和功能的研究进展。     

Argonaute蛋白与小RNA的作用机制

RNA干扰(RNA interference, RNAi)是在植物、动物、线虫、真菌以及昆虫等生物体中普遍存在的通过双链RNA(double strand RNA, dsRNA)诱导的抑制同源基因表达的一种保守的调控机制.小分子RNA通过特异性地识别结合RNA诱导的沉默复合体（RNA-induced silencing complex, RISC）对目标mRNA的表达在转录和翻译水平进行抑制.作为RISC的重要组成成分,Argonaute蛋白（Ago）发挥了至关重要的作用.为了进一步阐明Ago蛋白在RNA干扰中对小分子RNA的作用机制,本文介绍了Ago蛋白的结构、分类及其在RNA干扰机制中的作用,并着重阐述了目前已知的植物Ago蛋白对小分子RNA的几种作用机制,以及目前研究发现的Ago蛋白的功能作用,从而更进一步证实Ago蛋白对小分子RNA的作用是一个复杂的过程.     

Argonaute proteins: key players in RNA silencing

During the past decade, small non-coding RNAs have rapidly emerged as important contributors to gene regulation. To carry out their biological functions, these small RNAs require a unique class of proteins called Argonautes. The discovery and our comprehension of this highly conserved protein family is closely linked to the study of RNA-based gene silencing mechanisms. With their functional domains, Argonaute proteins can bind small non-coding RNAs and control protein synthesis, affect messenger RNA stability and even participate in the production of a new class of small RNAs, Piwi-interacting RNAs.     

RNA干涉在纤毛虫中的研究进展

RNA干涉是dsRNA介导的基因沉默现象,本文简要介绍了其作用的机制和生物学意义,重点阐述了RNA干涉在原生动物纤毛虫中的发现与应用,比较了RNA干涉与纤毛虫大核基因组重排机理的异同,并对RNA干涉在纤毛虫中传输的技术途径-RNAi喂饲法的原理也做了详细的介绍。     

Identification of eight members of the Argonaute family in the human genome

A number of genes have been identified as members of the Argonaute family in various nonhuman organisms and these genes are considered to play important roles in the development and maintenance of germ-line stem cells. In this study, we identified the human Argonaute family, consisting of eight members. Proteins to be produced from these family members retain a common architecture with the PAZ motif in the middle and Piwi motif in the C-terminal region. Based on the sequence comparison, eight members of the Argonaute family were classified into two subfamilies: the PIWI subfamily (PIWIL1/HIWI, PIWIL2/HILI, PIWIL3, and PIWIL4/HIWI2) and the eIF2C/AGO subfamily (EIF2C1/hAGO1, EIF2C2/hAGO2, EIF2C3/hAGO3, and EIF2C4/hAGO4). PCR analysis using human multitissue cDNA panels indicated that all four members of the PIWI subfamily are expressed mainly in the testis, whereas all four members of the eIF2C/AGO subfamily are expressed in a variety of adult tissues. Immunoprecipitation and affinity binding experiments using human HEK293 cells cotransfected with cDNAs for FLAG-tagged DICER, a member of the ribonuclease III family, and the His-tagged members of the Argonaute family suggested that the proteins from members of both subfamilies are associated with DICER. We postulate that at least some members of the human Argonaute family may be involved in the development and maintenance of stem cells through the RNA-mediated gene-quelling mechanisms associated with DICER.     

Argonaute: A scaffold for the function of short regulatory RNAs

Argonaute is the central protein component of RNA-silencing mechanisms. It provides the platform for target-mRNA recognition by short regulatory guide RNA strands and the Slicer catalytic activity for mRNA cleavage in RNA interference. Multiple Argonaute sub-families can be identified phylogenetically yet, despite this diversity, molecular and sequence analyses show that Argonaute proteins share common molecular properties and the capacity to function through a common mechanism. Recently, the members of the Piwi sub-family have been shown to interact with new classes of short regulatory RNAs, Piwi-interacting RNAs (piRNAs) and repeat-associated small interfering RNAs (rasiRNAs), which has implications for developmental processes and introduces a new dimension to the field of RNA silencing.     

Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells

Members of the Argonaute (Ago) protein family associate with small RNAs and have important roles in RNA silencing. Here, we analysed Ago1- and Ago2-containing protein complexes in human cells. Separation of Ago-associated messenger ribonucleoproteins (mRNPs) showed that Ago1 and Ago2 reside in three complexes with distinct Dicer and RNA-induced silencing complex activities. A comprehensive proteomic analysis of Ago-containing mRNPs identified a large number of proteins involved in RNA metabolism. By using co-immunoprecipitation experiments followed by RNase treatment, we biochemically mapped interactions within Ago mRNPs. Using reporter assays and knockdown experiments, we showed that the putative RNA-binding protein RBM4 is required for microRNA-guided gene regulation.     

Residues that affect human Argonaute2 concentration in cytoplasmic processing bodies

Sequence-specific gene silencing triggered by double-stranded RNA is a fundamental gene regulatory mechanism present in almost all eukaryotes. Argonaute2 (Ago2) is the central protein component of RNA-induced silencing complex (RISC), and resides in cytoplasmic processing bodies (P-bodies). In the present study, we demonstrated one human mutant Ago2 protein containing 6 point mutations (G32W, F128L, R196Q, P458S, T741A, S752G) failed to accumulate in P-bodies. Analysis of the different Ago2 revertants indicates the S752 as a key amino acid for P-body localization of Ago2. The S752 is evolutionary conserved in the Piwi domain of Ago2 homologs from worms, insects, plants and mammals. We further showed the single point mutation S752G interfering the interaction between Ago2 and Dcp1a, a key component of P-bodies.     

Identification and characterization of novel mouse and human ADAM33s with potential metalloprotease activity

The ADAM family of membrane-anchored proteins has a unique domain structure, with each containing a disintegrin and metalloprotease (ADAM) domain. We have isolated mouse and human cDNAs encoding a novel member of the ADAM family. The mouse and human predicted proteins consisted of 797 and 813 amino acids, respectively, and they shared 70% homology of the entire amino acid sequence. The mouse ADAM gene exists at a single gene locus. The human gene was ubiquitously expressed in tissues other than liver, was mapped to human chromosome 20p13, and was found to consist of 22 exons. Both proteins have domain organization identical to that of previously reported members of the ADAM family, and contain the typical zinc-binding consensus sequence (HEXGHXXGXXHD) in their metalloprotease domain and a pattern of cysteine localization (C(x)(3)C(x)(5)C(x)(5)CxC(x)(8)C) in their EGF-like domain that is typical of an EGF-like motif. The human protein shows homology with Xenopus ADAM13 (44%), human ADAM19 (40%), and human ADAM12 (39%). From the results of phylogenic analysis based on primary amino acid sequence and distribution of the mRNA, these novel ADAM genes were thus named ADAM33.     

Potential coexistence of both bacterial and eukaryotic small RNA biogenesis and functional related protein homologs in Archaea

RNA silencing plays crucial roles in both bacteria and eukaryotes, yet its machinery appears to differ in these two kingdoms. A couple of Argonaute protein homologs have been reported in some archaeal species in recent years. As Argonaute protein is the key component of eukaryotic RNA silencing pathways, such findings suggested the possibility of existence of eukaryotic RNA silencing like pathways in Archaea, which present the life forms between prokaryotes and eukaryotes. To further explore such hypothesis, we systematically screened 71 fully sequenced archaeal genomes, and identified some proteins containing homologous regions to the functional domains of eukaryotie RNA silencing pathway key proteins. The phylogenetic relationships of these proteins were analyzed. The conserved time-tional amino acids between archaeal and eukaryotic Piwi domains suggested their functional similarity. Our results provide new clues to the evolution of RNA silencing pathways.