RNA-induced Silencing Complex Activity Research Articles

Functional analysis of dicer-2 missense mutations in the siRNA pathway of Drosophila

The Drosophila RNase III enzyme Dicer-2 processes double-stranded RNA (dsRNA) precursors into small interfering RNAs (siRNAs). It also interacts with the siRNA product and R2D2 protein to facilitate the assembly of an RNA-induced silencing complex (RISC) that mediates RNA interference. Here, we characterized six independent missense mutations in the dicer-2 gene. Four mutations (P8S, L188F, R269W, and P365L) in the DExH helicase domain reduced dsRNA processing activity. Two mutations were located within an RNase III domain. P1496L caused a loss of dsRNA processing activity comparable to a null dicer-2 mutation. A1453T strongly reduced both dsRNA processing and RISC activity, and decreased the levels of Dicer-2 and R2D2 proteins, suggesting that this mutation destabilizes Dicer-2. We also found that the carboxyl-terminal region of R2D2 is essential for Dicer-2 binding. These results provide further insight into the structure–function relationship of Dicer, which plays a critical role in the siRNA pathway.

RNA Helicase A Interacts with RISC in Human Cells and Functions in RISC Loading

RNA interference is a conserved pathway of sequence-specific gene silencing that depends on small guide RNAs and the action of proteins assembled in the RNA-induced silencing complex (RISC). Minimally, the action of RISC requires the endonucleolytic slicer activity of Argonaute2 (Ago2) directed to RNA targets whose sequences are complementary to RISC-incorporated small RNA. To identify RISC components in human cells, we developed an affinity-purification strategy to isolate siRNA-programmed RISC. Here we report the identification of RNA helicase A (RHA) as a human RISC-associated factor. We show that RHA interacts in human cells with siRNA, Ago2, TRBP, and Dicer and functions in the RNAi pathway. In RHA-depleted cells, RNAi was reduced as a consequence of decreased intracellular concentration of active RISC assembled with the guide-strand RNA and Ago2. Our results identify RHA as a RISC component and demonstrate that RHA functions in RISC as an siRNA-loading factor.

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

Single-stranded small interfering RNA (siRNA) guides the cleavage of homologous mRNA by Argonaute proteins, the catalytic core of the RNA-induced silencing complex (RISC), in the conserved RNA interference (RNAi) pathway. The separation of the siRNA duplex into single strands is essential for the activation of RISC. Previous biochemical studies have suggested that Argonaute proteins cleave and remove the passenger strand of siRNA duplex from RISC, but the in vivo importance of this process and the mechanism for the removal of the nicked passenger strand are not known. Here, we show that in the filamentous fungus Neurospora, the Argonaute homolog QDE-2 and its slicer function are required for the generation of single-stranded siRNA and gene silencing in vivo. Biochemical purification of QDE-2 led to the identification of QIP, a QDE-2-interacting protein, with an exonuclease domain. The disruption of qip in Neurospora impaired gene silencing and siRNA accumulated, mostly in nicked duplex form. Furthermore, our results suggest that QIP acts as an exonuclease that cleaves and removes the nicked passenger strand from siRNA duplex in a QDE-2-dependent manner. Together, these results suggest that both the cleavage and removal of the passenger strand from the siRNA duplex are important steps in RNAi pathways.

Characterizing a Key Player in Micro, but Not Small, RNA Interference

Translation repression by RNA interference (RNAi) has emerged in recent years as a ubiquitous, powerful, and precise method for the regulation of gene expression, both naturally and experimentally. In this process, a messenger RNA (mRNA), made from a gene and destined to be translated into a protein, is silenced by a molecule of “interfering” RNA. At least two related processes fall under the heading of RNAi; in each, the interfering RNA is a small molecule (about 21 nucleotides long) that binds to a matching sequence on the mRNA. Exactly what happens next depends on how close the match is. Perfect complementarity (involving so-called small interfering RNAs, or siRNAs) results in mRNA degradation, while an imperfect match leads to silencing of the message without degradation. In this latter, non-degrading, form of interference, which has been widely observed in organisms from plants to people, the imperfectly complementary RNA is called a microRNA (miRNA), and is part of a large complex called the RISC (RNA-induced silencing complex). Neither the full structure of the RISC nor the details of its function are known, but several components have been identified, including the cleaving enzyme Dicer and the argonaute proteins Ago1 and Ago2. In the cytoplasm, after RISC binds to target mRNA, it accumulates in so-called P-bodies—poorly characterized multi-protein complexes that contain, among other things, the protein RCK/p54. In a new study, Chia-ying Chu and Tariq Rana demonstrate that, through its direct interactions with the argonaute proteins, and its promotion of P-body formation, RCK/p54 plays a key role in miRNA-mediated, but not siRNA-mediated, translation repression. The authors began by determining which P-body proteins can bind to which RISC proteins, using fluorescent tags to mark the various proteins. They found that Ago2 formed complexes with most of the known players. But they reasoned that the RNA scaffolding of the P-body likely causes many proteins to associate without actually binding to one another, and so, to rule out nonspecific effects, they digested the RNA out of the preparation. This left only RCK/p54 and Ago1 clinging to Ago2, indicating a true direct interaction. They next asked whether RCK/p54, first identified in P-bodies, was also a component of the active RISC. By purifying active complexes and analyzing their composition, they showed that it was—when a RISC was activated by the presence of a microRNA, it also contained RCK/p54. When the authors suppressed expression of RCK/p54 (neatly enough, by using an siRNA against it), P-bodies were disrupted, and Ago2 dispersed throughout the cytoplasm, suggesting RCK/p54 is responsible for the localization of Ago2 in P-bodies. P-bodies could also be disrupted by suppressing another protein, Lsm1; when this occurred, though, RCK/p54 still associated with Ago2 and with the RISC, indicating that these interactions do not depend on the presence of P-bodies. RCK/p54 depletion also provided the authors with another important insight: loss of RCK/p54 did not prevent siRNA-mediated RNA cleavage—the form of translation repression that depends on exact complementarity between an siRNA and mRNA. It did, however, lead to an increase in translation of known microRNA targets, consistent with a role for RCK/p54 in the second, non-degrading, form of translation repression, mediated by miRNA. Taken together, these results suggest that RCK/p54 forms part of the RISC in association with the argonaute proteins; that its previously identified association with P-bodies is a consequence of its function in the RISC; and that it plays a key role in miRNA-mediated translation repression. These results not only shed light on this important and poorly understood system for controlling gene expression, but also, because RCK/p54 appears to play no role in siRNA-mediated repression, they lay the groundwork for developing a detailed understanding of the similarities and differences between these two important pathways to RNAi.

Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

The relationship among nephrin, podocin, CD2AP, and α-actinin might not be a true ‘interaction’ in podocyte

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1 , NPHS2 , CD2AP , and ACTN4 , can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or α -actinin-4 mRNA by using reconstructed RNA interference vector – psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of α -actinin-4, whereas α -actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them.

A human, ATP-independent, RISC assembly machine fueled by pre-miRNA.

RNA interference (RNAi) is mediated by RNA-induced silencing complexes (RISCs), which are guided by microRNAs (miRNAs) or short interfering RNAs (siRNAs) to cognate RNA targets. In humans, the catalytic engine of RISC is a ribonucleoprotein formed by the Argonaute2 (Ago2) protein and either miRNA (miRNP) or siRNA (siRNP). The Dicer nuclease produces mature miRNAs and siRNAs from pre-miRNAs and double-stranded RNA (dsRNA), respectively, and associates with Ago2. Here, we studied the assembly of human RISC by presenting pre-miRNA to immunopurified complexes that contain Ago2, Dicer, and TRBP. Mature miRNAs were produced in an ATP-independent manner and guided specific cleavage of cognate RNA targets in a pattern that is typical of RISC. This de novo formed RISC activity dissociated from Dicer. The asymmetry of the RISC loading process was fully recapitulated in this system, which, however, could not efficiently assemble RISC from siRNA duplexes. Our findings demonstrate that, in humans, a miRNA loading complex (miRLC) is formed by Ago2 and Dicer prior to their encounter with pre-miRNA. We suggest that the miRLC couples the processing of the pre-miRNA substrate to the unwinding of the product and that after loading of the mature miRNA to Ago2, the miRLC disassembles and the miRNP is released.

Argonaute2 Cleaves the Anti-Guide Strand of siRNA during RISC Activation

The mRNA-cleavage step of RNA interference is mediated by an endonuclease, Argonaute2 (Ago2), within the RNA-induced silencing complex (RISC). Ago2 uses one strand of the small interfering (si) RNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone at a specific site measured from the guide strand's 5' end. Here, we show that both strands of siRNA get loaded onto Ago2 protein in Drosophila S2 cell extracts. The anti-guide strand behaves as a RISC substrate and is cleaved by Ago2. This cleavage event is important for the removal of the anti-guide strand from Ago2 protein and activation of RISC.

Suppression of RNA Interference by Adenovirus Virus-Associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.

Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity.

RNA interference is carried out by the small double-stranded RNA-induced silencing complex (RISC). The RISC-bound small RNA guides the RISC complex to identify and cleave mRNAs with complementary sequences. The proteins that make up the RISC complex and cleave mRNA have not been unequivocally defined. Here, we report the biochemical purification of RISC activity to homogeneity from Drosophila Schnieder 2 cell extracts. Argonaute 2 (Ago-2) is the sole protein component present in the purified, functional RISC. By using a bioinformatics method that combines sequence-profile analysis with predicted protein secondary structure, we found homology between the PIWI domain of Ago-2 and endonuclease V and identified potential active-site amino acid residues within the PIWI domain of Ago-2.

RISC Assembly Defects in the Drosophila RNAi Mutant armitage

The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes in wild-type and armi mutant ovary lysates suggests that armi mutants support early steps in the RNAi pathway but are defective in the production of active RNA-induced silencing complex (RISC), which mediates target RNA destruction in RNAi. Our results suggest that armi is required for RISC maturation.

Silence of the Genes: A Targeted Approach to the Suppression of Specific Genes in Human Disease Using Small Interfering RNA (siRNA)

The ability to silence specific genes of choice consistently and efficiently has always been a major goal for scientists. The emerging field of RNA interference (RNAi), a process in which target mRNAs are degraded by small interfering RNA (siRNA), may indeed provide this long sought after tool. The importance of this technology has been highlighted recently by Science, which has voted the RNAi discoveries as the “ & ;Breakthrough of the Year” in 2002. Essentially, RNAi involves an initiation and an effector step whereby introduced dsRNA is digested into 19-21 duplex siRNA by cleavage with Dicer and siRNA binds to an RNA-induced silencing complex (RISC). Activation of RISC targets the homologous sequence (transcript) and results in the cleavage of mRNA. The models of this RNAi mechanism and its applications, derived from biochemical and genetic approaches, are described in this minireview. So far, RNAi has proven to be a useful technique for genomic studies in C.elegans, D.melanogaster and various plants, for example. The use of RNAi has also been invaluable in studies in which morphological and developmental variability between species was investigated. Targeted, sequence specific siRNAs that suppress or silence gene expression have the potential to be in great demand as tools for for the treatment of human disease. There have already been several studies that have utilized siRNA to inhibit HIV-1 infection and replication, for example. The expansion of RNAi for biomedical therapeutics seems inevitable. This minireview analytically summarizes the advantages and the current and future potential of RNAi technology whilst simultaneously investigating any shortfalls or difficulties. Importantly, in vitro and in vivo applications in the laboratory and in human disease models are also described. Keywords: sirna, rnai, co-suppression, ptgs