Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production

RNA interference (RNAi) is now widely used for gene silencing in mammalian cells. The mechanism uses the RNA-induced silencing complex, in which Dicer, Ago2, and the human immunodeficiency virus type 1 (HIV-1) TAR RNA binding protein (TRBP) are the main components. TRBP is a protein that increases HIV-1 expression and replication by inhibition of the interferon-induced protein kinase PKR and by increasing translation of viral mRNA. After HIV infection, TRBP could restrict the viral RNA through its activity in RNAi or could contribute more to the enhancement of viral replication. To determine which function will be predominant in the virological context, we analyzed whether the inhibition of its expression could enhance or decrease HIV replication. We have generated small interfering RNAs (siRNAs) against TRBP and found that they decrease HIV-1 long terminal repeat (LTR) basal expression 2-fold, and the LTR Tat transactivated level up to 10-fold. In the context of HIV replication, siRNAs against TRBP decrease the expression of viral genes and inhibit viral production up to fivefold. The moderate increase in PKR expression and activation indicates that it contributes partially to viral gene inhibition. The moderate decrease in micro-RNA (miRNA) biogenesis by TRBP siRNAs suggests that in the context of HIV replication, TRBP functions other than RNAi are predominant. In addition, siRNAs against Dicer decrease viral production twofold and impede miRNA biogenesis. These results suggest that, in the context of HIV replication, TRBP contributes mainly to the enhancement of virus production and that Dicer does not mediate HIV restriction by RNAi.     

Differential roles of human Dicer-binding proteins TRBP and PACT in small RNA processing

During RNA interference and related gene regulatory pathways, the endonuclease Dicer cleaves precursor RNA molecules to produce microRNAs (miRNAs) and short interfering RNAs (siRNAs). Human cells encode a single Dicer enzyme that can associate with two different double-stranded RNA (dsRNA)-binding proteins, protein activator of PKR (PACT) and trans-activation response RNA-binding protein (TRBP). However, the functional redundancy or differentiation of PACT and TRBP in miRNA and siRNA biogenesis is not well understood. Using a reconstituted system, we show here that PACT and TRBP have distinct effects on Dicer-mediated dsRNA processing. In particular, we found that PACT in complex with Dicer inhibits the processing of pre-siRNA substrates when compared with Dicer and a Dicer–TRBP complex. In addition, PACT and TRBP show non-redundant effects on the production of different-sized miRNAs (isomiRs), which in turn alter target-binding specificities. Experiments using chimeric versions of PACT and TRBP suggest that the two N-terminal RNA-binding domains of each protein confer the observed differences in dsRNA substrate recognition and processing behavior of Dicer–dsRNA-binding protein complexes. These results support the conclusion that in humans, Dicer-associated dsRNA-binding proteins are important regulatory factors that contribute both substrate and cleavage specificity during miRNA and siRNA production.     

The role of PACT in the RNA silencing pathway

Small RNA-mediated gene silencing (RNA silencing) has emerged as a major regulatory pathway in eukaryotes. Identification of the key factors involved in this pathway has been a subject of rigorous investigation in recent years. In humans, small RNAs are generated by Dicer and assembled into the effector complex known as RNA-induced silencing complex (RISC) by multiple factors including hAgo2, the mRNA-targeting endonuclease, and TRBP (HIV-1 TAR RNA-binding protein), a dsRNA-binding protein that interacts with both Dicer and hAgo2. Here we describe an additional dsRNA-binding protein known as PACT, which is significant in RNA silencing. PACT is associated with an approximately 500 kDa complex that contains Dicer, hAgo2, and TRBP. The interaction with Dicer involves the third dsRNA-binding domain (dsRBD) of PACT and the N-terminal region of Dicer containing the helicase motif. Like TRBP, PACT is not required for the pre-microRNA (miRNA) cleavage reaction step. However, the depletion of PACT strongly affects the accumulation of mature miRNA in vivo and moderately reduces the efficiency of small interfering RNA-induced RNA interference. Our study indicates that, unlike other RNase III type proteins, human Dicer may employ two different dsRBD-containing proteins that facilitate RISC assembly.     

TRBP alters human precursor microRNA processing in vitro

MicroRNAs play central roles in controlling gene expression in human cells. Sequencing data show that many miRNAs are produced at different levels and as multiple isoforms that can vary in length at their 5′ or 3′ ends, but the biogenesis and functional significance of these RNAs are largely unknown. We show here that the human trans-activation response (TAR) RNA binding protein (TRBP), a known molecular partner of the miRNA processing enzyme Dicer, changes the rates of pre-miRNA cleavage in an RNA-structure-specific manner. Furthermore, TRBP can trigger the generation of iso-miRNAs (isomiRs) that are longer than the canonical sequence by one nucleotide. We show that this change in miRNA processing site can alter guide strand selection, resulting in preferential silencing of a different mRNA target. These results implicate TRBP as a key regulator of miRNA processing and targeting in humans.     

Structural basis of siRNA recognition by TRBP double‐stranded RNA binding domains

The accurate cleavage of pre‐micro(mi)RNAs by Dicer and mi/siRNA guide strand selection are important steps in forming the RNA‐induced silencing complex (RISC). The role of Dicer binding partner TRBP in these processes remains poorly understood. Here, we solved the solution structure of the two N‐terminal dsRNA binding domains (dsRBDs) of TRBP in complex with a functionally asymmetric siRNA using NMR, EPR, and single‐molecule spectroscopy. We find that siRNA recognition by the dsRBDs is not sequence‐specific but rather depends on the RNA shape. The two dsRBDs can swap their binding sites, giving rise to two equally populated, pseudo‐symmetrical complexes, showing that TRBP is not a primary sensor of siRNA asymmetry. Using our structure to model a Dicer‐TRBP‐siRNA ternary complex, we show that TRBP's dsRBDs and Dicer's RNase III domains bind a canonical 19 base pair siRNA on opposite sides, supporting a mechanism whereby TRBP influences Dicer‐mediated cleavage accuracy by binding the dsRNA region of the pre‐miRNA during Dicer cleavage.     

TAR RNA结合蛋白在HIV-1感染中的作用

TARRNA结合蛋白是细胞中双链RNA结合蛋白家族成员之一.它可以结合HIV-1TARRNA,并与Tat协同作用激活LTR表达,进而促进病毒的转录与翻译.TRBP也是将干扰素抗病毒通路与RNA干扰免疫通路相连的一种细胞蛋白.在干扰素诱生的PKR反应中,TRBP通过直接抑制PKR的自磷酸化、与PKR竞争通用的RNA底物或与PACT形成异源二聚体等机制抑制细胞内的PKR反应,从而降低了PKR介导的对病毒表达的抑制作用.TRBP与Dicer和Ago2等组成的RNA诱导沉默复合体,在RNA干扰中发挥着关键作用并调控随后的序列特异性降解.在HIV-1感染中,TRBP更倾向于促进病毒的表达与复制,因此TRBP也成为控制HIV-1感染的新靶点.     

Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.     

The Multiple Functions of TRBP,at the Hub of Cell Responses to Viruses,Stress, and Cancer

Summary: The TAR RNA binding protein (TRBP) has emerged as a key player in many cellular processes. First identified as a cellular protein that facilitates the replication of human immunodeficiency virus, TRBP has since been shown to inhibit the activation of protein kinase R (PKR), a protein involved in innate immune responses and the cellular response to stress. It also binds to the PKR activator PACT and regulates its function. TRBP also contributes to RNA interference as an integral part of the minimal RNA-induced silencing complex with Dicer and Argonaute proteins. Due to its multiple functions in the cell, TRBP is involved in oncogenesis when its sequence is mutated or its expression is deregulated. The depletion or overexpression of TRBP results in malignancy, suggesting that the balance of TRBP expression is key to normal cellular function. These studies show that TRBP is multifunctional and mediates cross talk between different pathways. Its activities at the molecular level impact the cellular function from normal development to cancer and the response to infections.     

TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing

Dicer is a key enzyme involved in RNA interference (RNAi) and microRNA (miRNA) pathways. It is required for biogenesis of miRNAs and small interfering RNAs (siRNAs), and also has a role in the effector steps of RNA silencing. Apart from Argonautes, no proteins are known to associate with Dicer in mammalian cells. In this work, we describe the identification of TRBP (human immunodeficiency virus (HIV-1) transactivating response (TAR) RNA-binding protein) as a protein partner of human Dicer. We show that TRBP is required for optimal RNA silencing mediated by siRNAs and endogenous miRNAs, and that it facilitates cleavage of pre-miRNA in vitro. TRBP had previously been assigned several functions, including inhibition of the interferon-induced double-stranded RNA-regulated protein kinase PKR and modulation of HIV-1 gene expression by association with TAR. The TRBP-Dicer interaction shown raises interesting questions about the potential interplay between RNAi and interferon-PKR pathways.     

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.     

The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing

Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid‐transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA‐induced silencing complexes), encounter of the target mRNA, and Ago2‐mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA‐ and siRNA‐loaded Ago2 populations co‐sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon‐induced protein kinase (PACT). Fractionation and membrane co‐immune precipitations further confirm that siRNA‐loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC‐associated double‐stranded siRNA, diagnostic of RISC loading, and RISC‐mediated mRNA cleavage products exclusively co‐sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA‐independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA‐mediated RNA silencing.     

HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA-binding protein, TRBP

TAR RNA-binding protein, TRBP, was recently discovered to be an essential partner for Dicer and a crucial component of the RNA-induced silencing complex (RISC), a critical element of the RNA interference (RNAi) of the cell apparatus. Human TRBP was originally characterized and cloned 15 years ago based on its high affinity for binding the HIV-1 encoded leader RNA, TAR. RNAi is used, in part, by cells to defend against infection by viruses. Here, we report that transfected TAR RNA can attenuate the RNAi machinery in human cells. Our data suggest that TAR RNA sequesters TRBP rendering it unavailable for downstream Dicer-RISC complexes. TAR-induced inhibition of Dicer-RISC activity in transfected cells was partially relieved by exogenous expression of TRBP.     

Oncogenic potential of TAR RNA binding protein TRBP and its regulatory interaction with RNA-dependent protein kinase PKR.

TAR RNA binding protein (TRBP) belongs to an RNA binding protein family that includes the double-stranded RNA-activated protein kinase (PKR), Drosophila Staufen and Xenopus xlrbpa. One member of this family, PKR, is a serine/threonine kinase which has anti-viral and anti-proliferative effects. In this study we show that TRBP is a cellular down-regulator of PKR function. Assaying expression from an infectious HIV-1 molecular clone, we found that PKR inhibited viral protein synthesis and that over-expression of TRBP effectively countered this inhibition. In intracellular and in cell-free assays we show that TRBP directly inhibits PKR autophosphorylation through an RNA binding-independent pathway. Biologically, TRBP serves a growth-promoting role; cells that overexpress TRBP exhibit transformed phenotypes. Our results demonstrate the oncogenic potential of TRBP and are consistent with the notion that intracellular PKR function contributes physiologically towards regulating cellular proliferation.     

Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA

Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.     

dsRNA binding properties of RDE-4 and TRBP reflect their distinct roles in RNAi

Double-stranded RNA (dsRNA)-binding proteins facilitate Dicer functions in RNA interference. Caenorhabditis elegans RDE-4 facilitates cleavage of long dsRNA to small interfering RNA (siRNA), while human trans-activation response RNA-binding protein (TRBP) functions downstream to pass siRNA to the RNA-induced silencing complex. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based on the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds noncooperatively. Our studies offer a paradigm for how dsRNA-binding proteins, which are not sequence specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRNA-binding motif 2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation.