Human RNA-induced Silencing Complex Research Articles

XRN2 suppresses aberrant entry of tRNA trailers into argonaute in humans and Arabidopsis.

MicroRNAs (miRNAs) are a well-characterized class of small RNAs (sRNAs) that regulate gene expression post-transcriptionally. miRNAs function within a complex milieu of other sRNAs of similar size and abundance, with the best characterized being tRNA fragments or tRFs. The mechanism by which the RNA-induced silencing complex (RISC) selects for specific sRNAs over others is not entirely understood in human cells. Several highly expressed tRNA trailers (tRF-1s) are strikingly similar to microRNAs in length but are generally excluded from the microRNA effector pathway. This exclusion provides a paradigm for identifying mechanisms of RISC selectivity. Here, we show that 5' to 3' exoribonuclease XRN2 contributes to human RISC selectivity. Although highly abundant, tRF-1s are highly unstable and degraded by XRN2 which blocks tRF-1 accumulation in RISC. We also find that XRN mediated degradation of tRF-1s and subsequent exclusion from RISC is conserved in plants. Our findings reveal a conserved mechanism that prevents aberrant entry of a class of highly produced sRNAs into Ago2.

Partial reconstitution of the RNAi response in human cells using Drosophila gene products.

While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two cofactors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR. We observed significant production of ∼21-nt long siRNAs, derived from a cotransfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. Surprisingly, dDcr2 was able to produce siRNAs even in the absence of Loqs-PD, which is thought to be required for dsRNA cleavage by dDcr2. This result may be explained by our finding that dDcr2 is able to bind the human Loqs-PD homolog TRBP when expressed in human cells in the absence of Loqs-PD. We conclude that it is possible to at least partially rescue the ability of mammalian somatic cells to express functional siRNAs using gene products of invertebrate origin.

Slicer-independent mechanism drives small-RNA strand separation during human RISC assembly.

Small RNA silencing is mediated by the effector RNA-induced silencing complex (RISC) that consists of an Argonaute protein (AGOs 1–4 in humans). A fundamental step during RISC assembly involves the separation of two strands of a small RNA duplex, whereby only the guide strand is retained to form the mature RISC, a process not well understood. Despite the widely accepted view that ‘slicer-dependent unwinding’ via passenger-strand cleavage is a prerequisite for the assembly of a highly complementary siRNA into the AGO2-RISC, here we show by careful re-examination that ‘slicer-independent unwinding’ plays a more significant role in human RISC maturation than previously appreciated, not only for a miRNA duplex, but, unexpectedly, for a highly complementary siRNA as well. We discovered that ‘slicer-dependency’ for the unwinding was affected primarily by certain parameters such as temperature and Mg2+. We further validate these observations in non-slicer AGOs (1, 3 and 4) that can be programmed with siRNAs at the physiological temperature of humans, suggesting that slicer-independent mechanism is likely a common feature of human AGOs. Our results now clearly explain why both miRNA and siRNA are found in all four human AGOs, which is in striking contrast to the strict small-RNA sorting system in Drosophila.

Designing Ago2-specific siRNA/shRNA to Avoid Competition with Endogenous miRNAs.

Relatively large amounts of transfected siRNA can compete for Ago proteins and thus compromise endogenous miRNA function, potentially leading to toxicities. Here, we show that shRNA can also perturb endogenous miRNA function similarly. More importantly, we also show that the problem can be solved by designing shRNAs in the context of pre-miR-451 structure with completely complementary stem, which significantly improves the Ago2 specificity. This shRNA was shown to be Ago2-specific, and maintain target-silencing ability while avoiding competition with endogenous miRNAs by not competing for Agos 1, 3, and 4. We conclude that modified pre-miR-451 structure provides a general platform to design shRNAs that significantly reduce perturbation of miRNA function.

Akt-Mediated Phosphorylation of Argonaute 2 Downregulates Cleavage and Upregulates Translational Repression of MicroRNA Targets

A high-throughput RNA interference (RNAi) screen targeting 542 genes of the human kinome was usedto discover regulators of RNAi. Here we report that the proto-oncogene Akt-3/PKBγ (Akt3) phosphorylates Argonaute 2 (Ago2) at S387, which downregulates cleavage and upregulates translational repression of endogenous microRNA (miRNA)-targeted messenger RNAs (mRNAs). We further demonstrate that Akt3 coimmunoprecipitates with Ago2 and phosphorylation of Ago2 at S387 facilitates its interaction with GW182 and localization to cytoplasmic processing bodies (P bodies), where miRNA-targeted mRNAs are thought to be stored and degraded. Therefore, Akt3-mediated phosphorylation of Ago2 is a molecular switch between target mRNA cleavage and translational repression activities of Ago2.

MicroRNA Factory: RISC Assembly from Precursor MicroRNAs

RNA silencing requires assembly of an effector complex, RNA-induced silencing complex (RISC), composed of a small RNA and the Ago protein. In this issue of Molecular Cell, Liu et al. (2012) provide insights of miRNP/RISC assembly pathways in mammals, using an in vitro human RISC assembly assay programmed by pre-miRNAs.

Dissecting the interactions between Human Argonaute proteins and RISC components

MicroRNAs (miRNAs) are a group of non‐coding RNAs, with ~20–24 nt in length, that regulate gene expression at post‐transcriptional level. In human cells, miRNAs are loaded to Argonaute proteins (AGOs), which interact with other proteins to form RNA‐induced silencing complex (RISC). miRNAs then guide RISC to recognize their target mRNAs. These miRNA‐RNA‐protein complexes will trans‐locate to a specific cytoplasmic compartment called RNA processing body (P‐body), for the mRNA storage or degradation. It has been revealed that RCK/p54 (DDX6) is the key protein for P‐body formation and it also interacts with AGO2. Nevertheless, how RCK/p54 interacts with AGO2 to facilitate the miRNA‐mediate gene regulation in P‐body remains unclear. Here we report the domains and residues of AGO2 required for interacting with RCK/p54. Our preliminary data shows that the localization of AGO2 at various cytoplasmic RNA granules and the interaction with RCK/p54 are determined by different domains of AGO2, suggesting that miRNA‐mediated translational repression is unrelated to the miRISC localization at P‐body. Our continuing analysis of AGO2 deletion mutants interacting with other RISC components will have important implications for the miRNA‐mediated gene silencing.

Structure of C3PO and mechanism of human RISC activation.

Assembly of the RNA-induced silencing complex (RISC) consists of loading duplex (guide/passenger) siRNA onto and removing the passenger strand from Argonaute (Ago2). Ago2 contributes critically to RISC activation by nicking the passenger strand. Here, we reconstituted duplex siRNA-initiated RISC activity using recombinant human (h)Ago2 and C3PO, indicating a critical role for C3PO in hAgo2-RISC activation. Consistently, genetic depletion of C3PO compromised RNA silencing in mammalian cells. We determined the crystal structure of hC3PO, which reveals an asymmetric octamer barrel consisting of six Translin and two TRAX subunits. This asymmetric assembly is critical for the function of C3PO as a novel endonuclease that cleaves RNA at the interior surface. The current work supports a Dicer-independent mechanism for human RISC activation: 1) Ago2 directly binds duplex siRNA and nicks the passenger strand; 2) C3PO activates RISC by degrading Ago2-nicked passenger strand.

Viral RNAi Suppressor Reversibly Binds siRNA to Outcompete Dicer and RISC via Multiple Turnover

RNA interference is a conserved gene regulatory mechanism employed by most eukaryotes as a key component of their innate immune response to viruses and retrotransposons. During viral infection, the RNase-III-type endonuclease Dicer cleaves viral double-stranded RNA into small interfering RNAs (siRNAs) 21–24 nucleotides in length and helps load them into the RNA-induced silencing complex (RISC) to guide the cleavage of complementary viral RNA. As a countermeasure, many viruses have evolved viral RNA silencing suppressors (RSS) that tightly, and presumably quantitatively, bind siRNAs to thwart RNA-interference-mediated degradation. Viral RSS proteins also act across kingdoms as potential immunosuppressors in gene therapeutic applications. Here we report fluorescence quenching and electrophoretic mobility shift assays that probe siRNA binding by the dimeric RSS p19 from Carnation Italian Ringspot Virus, as well as by human Dicer and RISC assembly complexes. We find that the siRNA:p19 interaction is readily reversible, characterized by rapid binding [(1.69±0.07)×108 M−1 s−1] and marked dissociation (koff=0.062±0.002 s−1). We also observe that p19 efficiently competes with recombinant Dicer and inhibits the formation of RISC-related assembly complexes found in human cell extract. Computational modeling based on these results provides evidence for the transient formation of a ternary complex between siRNA, human Dicer, and p19. An expanded model of RNA silencing indicates that multiple turnover by reversible binding of siRNAs potentiates the efficiency of the suppressor protein. Our predictive model is expected to be applicable to the dosing of p19 as a silencing suppressor in viral gene therapy.

ATP-dependent human RISC assembly pathways

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

Molecular Basis for Target RNA Recognition and Cleavage by Human RISC

The RNA-Induced Silencing Complex (RISC) is a ribonucleoprotein particle composed of a single-stranded short interfering RNA (siRNA) and an endonucleolytically active Argonaute protein, capable of cleaving mRNAs complementary to the siRNA. The mechanism by which RISC cleaves a target RNA is well understood, however it remains enigmatic how RISC finds its target RNA. Here, we show, both in vitro and in vivo, that the accessibility of the target site correlates directly with the efficiency of cleavage, demonstrating that RISC is unable to unfold structured RNA. In the course of target recognition, RISC transiently contacts single-stranded RNA nonspecifically and promotes siRNA-target RNA annealing. Furthermore, the 5' part of the siRNA within RISC creates a thermodynamic threshold that determines the stable association of RISC and the target RNA. We therefore provide mechanistic insights by revealing features of RISC and target RNAs that are crucial to achieve efficiency and specificity in RNA interference.

MicroRNA silencing through RISC recruitment of eIF6

MicroRNAs (miRNAs) are a class of small RNAs that act post-transcriptionally to regulate messenger RNA stability and translation. To elucidate how miRNAs mediate their repressive effects, we performed biochemical and functional assays to identify new factors in the miRNA pathway. Here we show that human RISC (RNA-induced silencing complex) associates with a multiprotein complex containing MOV10--which is the homologue of Drosophila translational repressor Armitage--and proteins of the 60S ribosome subunit. Notably, this complex contains the anti-association factor eIF6 (also called ITGB4BP or p27BBP), a ribosome inhibitory protein known to prevent productive assembly of the 80S ribosome. Depletion of eIF6 in human cells specifically abrogates miRNA-mediated regulation of target protein and mRNA levels. Similarly, depletion of eIF6 in Caenorhabditis elegans diminishes lin-4 miRNA-mediated repression of the endogenous LIN-14 and LIN-28 target protein and mRNA levels. These results uncover an evolutionarily conserved function of the ribosome anti-association factor eIF6 in miRNA-mediated post-transcriptional silencing.

Short Interfering RNA Strand Selection Is Independent of dsRNA Processing Polarity during RNAi in Drosophila

Short interfering RNAs (siRNAs) guide mRNA cleavage during RNA interference (RNAi). Only one siRNA strand assembles into the RNA-induced silencing complex (RISC), with preference given to the strand whose 5' terminus has lower base-pairing stability. In Drosophila, Dcr-2/R2D2 processes siRNAs from longer double-stranded RNAs (dsRNAs) and also nucleates RISC assembly, suggesting that nascent siRNAs could remain bound to Dcr-2/R2D2. In vitro, Dcr-2/R2D2 senses base-pairing asymmetry of synthetic siRNAs and dictates strand selection by asymmetric binding to the duplex ends. During dsRNA processing, Dicer (Dcr) liberates siRNAs from dsRNA ends in a manner dictated by asymmetric enzyme-substrate interactions. Because Dcr-2/R2D2 is unlikely to sense base-pairing asymmetry of an siRNA that is embedded within a precursor, it is not clear whether processed siRNAs strictly follow the thermodynamic asymmetry rules or whether processing polarity can affect strand selection. We use a Drosophila in vitro system in which defined siRNAs with known asymmetry can be generated from longer dsRNA precursors. These dsRNAs permit processing specifically from either the 5' or the 3' end of the thermodynamically favored strand of the incipient siRNA. Combined dsRNA-processing/mRNA-cleavage assays indicate that siRNA strand selection is independent of dsRNA processing polarity during Drosophila RISC assembly in vitro.

1023. New Generation of shRNA for Targeted Cancer Gene Therapy

Small interfering RNA (siRNA) molecules are powerful mediators for post-transcrptional gene silencing known as RNA interference. Short hairpin (shRNA) encoded within an expression vector can effectively harnessing the RNA interference process in mammalian cells. RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Argonaute2 (Ago2) has been found to be the catalytic endonuclease of human RISC that mediates cleavage of targeted mRNA. Interestingly, the other Argonaute family of proteins (Ago1, Ago3, Ago4) lack of endonuclease activity, but are nonetheless capable of loaded with single-stranded guide RNAs. RISC assembled with Ago1, Ago3, or Ago4 may be cleavage-independent pathway for which mediates translational repressions. Translational repression via sequestration of mRNA in the P-body is proposed to be the major gene silencing pathway for micro RNAs (miRNAs). We are actively developing targeted cancer gene therapy strategy through the application of shRNAs. In the past, siRNA and shRNA design strategies have been focused on the RNA degradation pathway (Ago2 cleavage-dependent). In this study, we seek to expand the shRNA design strategy to simultaneously mediate targeted RNA degradation and translational repression. Simultaneously mediate RNA degradation and translational repression is predicted to produce much quicker and sustained knock-down effect on targeted mRNA. We will present data to support our novel shRNA design strategy, examine the efficacy of novel shRNA design process and compare knock-down efficiency with conventional shRNA molecules.