RISC-loading Complex Research Articles

Methods for quantification and characterization of microRNAs in cell-free plasma/serum, normal exosomes and tumor-derived exosomes

Exosomes are actively released by all cell types under both normal and pathological conditions. The circulation of these membrane nanovesicles in various body fluids enables them to horizontally transfer their genetic information from cell to cell. An excessively high exosome secretion has been observed in cancer patients. Packaging of biomolecules, e.g., nucleic acids and proteins, into exosomes is assumed to be a selective process during tumor progression. Exosomes even contain microRNAs (miRNAs) associated with the RISC-Loading Complex, and thus, display cell-independent capacity to process precursor into mature miRNAs. These small non-coding RNA molecules are frequently deregulated in cancer and modulate the expression of numerous tumor-associated genes and cellular processes. Considering the characteristics of miRNAs and exosomes that reflect cancer development, tumor load, malignant progression towards metastatic relapse and drug resistance, they may be of potential clinical uses. To reach the high level of evidence required for their entry into clinical practice, it is crucial to develop standardized detection assays. The present review article focuses on most popular analytical techniques for isolating, quantifying and characterizing exosomes and miRNAs from plasma/serum of cancer patients, to end with an example for the fractionation of cancer-derived exosomal, normal exosomal and cell-free miRNAs.

Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro.

The human TAR RNA-binding protein (hTRBP) and protein activator of protein kinase R (hPACT) are important players in RNA interference (RNAi). Together with hArgonaute2 (hAgo2) and hDicer they have been reported to form the RISC-loading complex (RLC). Among other functions, hTRBP was suggested to assist the loading of hAgo2 with small interfering RNAs (siRNAs) within the RLC. Although several studies have been conducted to evaluate the specific functions of hTRBP and hPACT in RNAi, exact mechanisms and modes of action are still unknown. Here, we present a biochemical study further evaluating the role of hTRBP and hPACT in hAgo2-loading. We found that both proteins enhance hAgo2-mediated RNA cleavage significantly; even a hAgo2 mutant impaired in siRNA binding shows full cleavage activity in the presence of hTRBP or hPACT. Pre-steady state binding studies reveal that the assembly of wildtype-hAgo2 (wt-hAgo2) and siRNAs remains largely unaffected, whereas the binding of mutant hAgo2-PAZ9 to siRNA is restored by adding either hTRBP or hPACT. We conclude that both proteins assist in positioning the siRNA within hAgo2 to ensure optimal binding and cleavage. Overall, our data indicate that hTRBP and hPACT are part of a regulative system of RNAi that is important for efficient target RNA cleavage.

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies.

The Dicer-TRBP Interface Structure and Implications for Strand Selection During Microrna Biogenesis

Central to the human microRNA (miRNA) pathway of gene regulation is the RNA-induced silencing complex (RISC), which is composed of an Argonaute protein bound to a guide strand of single stranded RNA that grants specificity via complementarity to the gene target. The assembly of this ribonucleoprotein depends on the RISC-loading complex (RLC), which comprises the endoribonuclease Dicer, a dsRNA binding protein such as TRBP, and an Argonaute protein.We currently lack mechanistic details regarding the role of TRBP and its homologs in the process of RISC loading. TRBP is known to increase the stability of Dicer and promote interactions between the three protein components of the RLC. An additional hypothesized role for TRBP is to act as a chaperone that shuttles an RNA duplex from Dicer to Argonaute, potentially taking part in the process of strand selection that determines which arm of the double stranded RNA (dsRNA) will be loaded into the RISC to become the guide strand and thus which genes will be targeted.TRBP contains two canonical dsRNA binding domains (dsRBDs) known to bind dsRNA tightly, and a third non-canonical dsRBD that binds instead to Dicer. We used NMR spectroscopy to demonstrate that these three domains do not interact with each other. Using X-ray crystallography, we have determined the structure of the Dicer-TRBP interface, revealing where and how TRBP is anchored in the context of the RLC. Subsequent cellular experiments disrupted this Dicer-TRBP interaction to examine the effects of an absent TRBP on the miRNA processing pathway. Together these results shed light on TRBP's role in the poorly understood processes of RISC-loading and strand selection.

Arginine rich short linear motif of HIV-1 regulatory proteins inhibits Dicer dependent RNA interference

BackgroundArginine Rich Motif (ARM) of HIV-1 Tat and Rev are extensively studied linear motifs (LMs). They are already established as an inefficient bipartite nuclear localisation signal (NLS). The unusual passive diffusion of HIV-1 NLS tagged reporter proteins across the nucleus is due to an unknown competing functionality of ARM. Recent findings about the role of retroviral proteins as a suppressor of RNA interference (RNAi) involving their basic residues hint an interesting answer to this alternate functionality. The present work explores the role of HIV-1 ARM as a uniquely evolved viral motif to combat Dicer dependent RNAi.ResultsWe show that RNA binding ARM of both HIV-1 Tat and Rev is a LM with a pattern RXXRRXRRR unique to viruses. Extending the in silico results to wet lab, we proved both HIV-1 Tat and Rev can suppress Dicer dependent RNA silencing process involving ARM. We show, HIV-1 Tat and Rev and their corresponding ARM can bind the RISC loading complex (RLC) components TRBP and PACT confirming ARM as an independent RNAi suppression motif. Enhancement of RNAi in infection scenario through enoxacin increases HIV-1 replication as indicated by p24 levels. Except Dicer, all other cytoplasmic RNAi components enhance HIV-1 replication, indicating crucial role of Dicer independent (Ago2 dependent) RNAi pathway in HIV-1 infection. Sequence and structural analysis of endo/exo-microRNA precursors known to be regulated in HIV-1 infection highlights differential features of microRNA biogenesis. One such set of miRNA is viral TAR encoded HIV-1-miR-TAR-5p (Tar1) and HIV-1-miR-TAR-3p (Tar2) that are known to be present throughout the HIV-1 life cycle. Our qPCR results showed that enoxacin increases Tar2 miRNA level which is interesting as Tar2 precursor shows Ago2 dependent processing features.ConclusionsWe establish HIV-1 ARM as a novel viral motif evolved to target the Dicer dependent RNAi pathway. The conservation of such motif in other viral proteins possibly explains the potent suppression of Dicer dependent RNAi. Our model argues that HIV-1 suppress the processing of siRNAs through inhibition of Dicer while at the same time manipulates the RNAi machinery to process miRNA involved in HIV-1 replication from Dicer independent pathways.

Intracellular Localization and Routing of miRNA and RNAi Pathway Components

Several different pathways, generally termed RNA silencing pathways, utilize small RNA molecules guiding sequence-specific silencing effects of ribonucleoprotein effector complexes, traditionally termed RNA-induced silencing complex (RISC). Three RNA silencing pathways were recognized in mammalian cells: RNA interference (RNAi), where short RNAs produced from long double-stranded RNA guide cleavage of cognate mRNAs, microRNA (miRNA) pathway, where endogenously-encoded miRNAs typically induce translational repression, and piRNA pathway, where piRNAs (PIWI-associated RNAs) guide repression of repetitive sequences in the germline. Originally, RNAi and miRNA pathways were thought to act in the cytoplasm, however, there is a growing body of evidence that these pathways also have a nuclear component. This text reviews the current evidence concerning nuclear localization and function of miRNA and RNAi pathway components. We provide evidence that TRBP, Dicer and AGO2, proteins found in the RISC-loading complex (RLC) and RISC itself, are present in the nucleus. Nonetheless, fully functional RLC is not found in the nuclear compartment which is consistent with the recent findings obtained by Fluorescence Cross-Correlation Spectroscopy experiments illustrating that RISC is specifically loaded within the cytoplasm and shuttles subsequently between the nuclear and cytoplasmic compartment, thereby allowing small RNA gene regulation in both compartments. The function of nuclear TRBP and Dicer proteins remains elusive. We also discuss the consequences of nucleotide analogs introduced into siRNAs which can severely interfere with the natural cytoplasmic localization mediated by Exportin-5 which is required for efficient RISC loading in the cytoplasm.

Minor-Groove-Modulating Adenosine Replacements Control Protein Binding and RNAi Activity in siRNAs

Short-interfering RNAs (siRNAs) are common tools in molecular biology; however, the development of RNAi-based therapeutics is limited by immunostimulatory and nonspecific effects mediated by off-target RNA-binding proteins. The RNA-dependent protein kinase (PKR) and adenosine deaminase that acts on RNA 1 (ADAR1) are two proteins implicated in RNAi off-target effects and share a common means of interaction with siRNAs through double-stranded RNA binding motifs (dsRBMs). Here we report the site-specific introduction of N²-propargyl 2-aminopurine into siRNAs and subsequent conversion to two bulky products via copper-catalyzed azide alkyne cycloaddition (CuAAC) with either N-azidoacetyl-mannosamine azide or N-ethylpiperidine azide. We observed position-specific effects on RNAi activity for modifications made to both the passenger and guide strands. These findings are rationalized in light of recent structural studies of components of the RNA-induced silencing complex (RISC) and RISC-loading complex (RLC). The most active siRNAs were assayed for binding affinity to PKR and ADAR1. PKR binding was significantly reduced by multiple modifications, regardless of size, and ADAR1 binding was reduced in a position- and size-sensitive manner. Our findings present a strategy for designing siRNAs that reduce off-target dsRBM-protein binding while retaining native RNAi activity.

696 Regulation of MMP-13 (collagenase-3) by the transcription factor Pit-1

Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies.

Structural insights into RNA processing by the human RISC-loading complex

Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (siRNA or miRNA) into an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (Ago2) assembles with the guide RNA-generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC) necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to Ago2. Here we show, using single-particle electron microscopy analysis, that human Dicer exhibits an L-shaped structure. Withn the RLC Dicer's N-terminal DExH/D domain, located at the short base branch, interacts with TRBP, while its C-terminal catalytic domains in the main body are proximal to Ago2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to Ago2.

Off-target and a portion of target-specific siRNA mediated mRNA degradation is Ago2 ‘Slicer’ independent and can be mediated by Ago1

It is known that siRNAs are capable of reducing expression of non-target genes due to the interaction of the siRNA guide strand with a partially complementary site on the ‘off-target’ mRNA. In the current study, we show that reduction of cellular Ago2 levels has no effect on off-target reduction of endogenous genes and that off-target degradation of mRNA can occur even in an Ago2 knockout cell line. Using antisense mediated reduction of Ago proteins and chemically modified cleavage- and binding-deficient siRNAs, we demonstrate that siRNA mediated off-target reduction is Ago2 cleavage independent, but does require siRNA interaction with either Ago1 or Ago2 and the RISC-loading complex. We also show that depletion of P-body associated proteins results in a reduction of off-target siRNA-mediated degradation of mRNA. Finally, we present data suggesting that a significant portion of on-target siRNA activity is also Ago2 cleavage independent, however, this activity does not appear to be P-body associated.

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.

Recapitulation of Short RNA-Directed Translational Gene Silencing In Vitro

microRNAs (miRNAs) are a large class of endogenous short RNAs that repress gene expression. Many miRNAs are conserved throughout evolution, and dysregulation of miRNA pathways has been correlated with an increasing number of human diseases. In animals, miRNAs typically bind to the 3' untranslated region (3'UTR) of target mRNAs with imperfect sequence complementarity and repress translation. Despite their importance in regulating biological processes in numerous organisms, the mechanisms of miRNA function are largely unknown. Here, we report in vitro reactions for miRNA-directed translational gene silencing. These reactions faithfully recapitulate known in vivo hallmarks of mammalian miRNA function, including a requirement for a 5' phosphate and perfect complementarity to the mRNA target in the 5' seed region. Translational gene silencing by miRNAs in vitro requires target mRNAs to possess a 7-methyl G cap and a polyA tail, whereas increasing polyA tail length alone can increase miRNA silencing activity.

Molecular Requirements for RNA-induced Silencing Complex Assembly in the Drosophila RNA Interference Pathway

Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.