The RNA-binding region of human TRBP interacts with microRNA precursors through two independent domains.
ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression through RNA interference. Human miRNAs are generated through a series of enzymatic processing steps. The precursor miRNA (pre-miRNA) is recognized and cleaved by a complex containing Dicer and several non-catalytic accessory proteins. HIV TAR element binding protein (TRBP) is a constituent of the Dicer complex, which augments complex stability and potentially functions in substrate recognition and product transfer to the RNA-induced silencing complex. Here we have analysed the interaction between the RNA-binding region of TRBP and an oncogenic human miRNA, miR-155, at different stages in the biogenesis pathway. We show that the region of TRBP that binds immature miRNAs comprises two independent double-stranded RNA-binding domains connected by a 60-residue flexible linker. No evidence of contact between the two double-stranded RNA-binding domains was observed either in the apo- or RNA-bound state. We establish that the RNA-binding region of TRBP interacts with both pre-miR-155 and the miR-155/miR-155* duplex through the same binding surfaces and with similar affinities, and that two protein molecules can simultaneously interact with each immature miRNA. These data suggest that TRBP could play a role before and after processing of pre-miRNAs by Dicer.
Project description:MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.
Project description:MicroRNAs (miRNAs) mediate translational repression or degradation of their target messenger RNAs by RNA interference (RNAi). The primary transcripts of miRNA genes (pri-miRNAs) are sequentially processed by the nuclear Drosha-DGCR8 complex to approximately 60-70 nucleotide (nt) intermediates (pre-miRNAs) and then by the cytoplasmic Dicer-TRBP complex to approximately 20-22 nt mature miRNAs. Certain pri-miRNAs are subject to RNA editing that converts adenosine to inosine (A --> I RNA editing); however, the fate of edited pri-miRNAs is mostly unknown. Here, we provide evidence that RNA editing of pri-miR-151 results in complete blockage of its cleavage by Dicer and accumulation of edited pre-miR-151 RNAs. Our results indicate that A --> I conversion at two specific positions of the pre-miRNA foldback structure can affect its interaction with the Dicer-TRBP complex, showing a new regulatory role of A --> I RNA editing in miRNA biogenesis.
Project description: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.
Project description:We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr co-factor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its co-factors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs versus passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells. Series includes three datasets of total small RNA reads from wild type and Dicer negative 293T cells. Also included are total small RNA reads of the Dicer-negative cell line NoDice(4-25) transfected with a vector expressing human Dicer, Drosophila Dicer1, or mock transfected. RISC-associated small RNAs identified by ribonucleoprotein immunoprecipitation (RIP-Seq) in wild type 293T, Dicer-negative NoDice(4-25) line, and NoDice(4-25) transfected with either hsa-miR-155 or kshv-miR-K12-11 miRNA duplexes. The final data series are PAR-CLIP libraries which identified microRNA targets in untransfected NoDice(4-25) cells and NoDice(4-25) cells transfected with either hsa-miR-92a, hsa-miR-155, kshv-miR-K12-11 miRNA duplexes
Project description:The RNA-binding protein TRBP is a central component of the Dicer complex. Despite a decade of biochemical and structural studies, the essential functionality of TRBP in microRNA (miRNA) biogenesis remains unknown. Here we show that TRBP is an integral cofactor for time-efficient Dicer processing in RNA-crowded environments. We competed for Dicer processing of pre-miRNA with a large amount of cellular RNA species and found that Dicer-TRBP, but not Dicer alone, remains resilient. To apprehend the mechanism of this substrate selectivity, we use single-molecule fluorescence. The real-time observation reveals that TRBP acts as a gatekeeper, precluding Dicer from engaging with pre-miRNA-like substrates. TRBP acquires the selectivity using the PAZ domain of Dicer, whereas Dicer moderates the RNA-binding affinity of TRBP for fast turnover. This coordinated action between TRBP and Dicer accomplishes an efficient way of discarding pre-miRNA-like substrates.
Project description:Drosophila Dicer-1 produces microRNAs (miRNAs) from pre-miRNA, whereas Dicer-2 generates small interfering RNAs (siRNAs) from long dsRNA. loquacious (loqs) encodes three Dicer partner proteins, Loqs-PA, Loqs-PB, and, Loqs-PD, generated by alternative splicing. To understand the function of each Loqs isoform, we constructed loqs isoform-specific rescue flies. Loqs-PD promotes siRNA production in vivo by Dicer-2. Loqs-PA or Loqs-PB is required for viability, but the proteins are not fully redundant: Loqs-PB is required to produce a specific subset of miRNAs. Surprisingly, Loqs-PB tunes the product size cleaved by Dicer-1 from pre-miR-307a, generating a longer miRNA isoform with a distinct seed sequence and target specificity. The mouse and human Dicer-binding partner TRBP, a homolog of Loqs-PB, similarly tunes the site of pre-miR-132 cleavage by mammalian Dicer. Thus, Dicer-binding partner proteins can change the choice of cleavage site by Dicer, producing miRNAs with different target specificities than those that would be made by Dicer alone. Examination of Dicer-binding proteins on small RNA profiles of female fly heads, fly ovaries, mouse embryonic fibroblasts, and mouse tail fibroblasts.
Project description: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.
Project description:BACKGROUND: The human TAR RNA-binding protein, TRBP, was first identified and cloned based on its high affinity binding to the small hairpin trans-activation responsive (TAR) RNA of HIV-1. TRBP has more recently been found to be a constituent of the RNA-induced silencing complex (RISC) serving as a Dicer co-factor in the processing of the ~70 nucleotide pre-microRNAs(miRNAs) to 21-25 nucleotide mature miRNAs. FINDINGS: Using co-immunoprecipitation and protein-identification by mass spectrometry, we characterized intracellular proteins that complex with TRBP. These interacting proteins include those that have been described to act in protein synthesis, RNA modifications and processing, DNA transcription, and cell proliferation. CONCLUSIONS: Our findings provide a proteome of factors that may cooperate with TRBP in activities such as miRNA processing and in RNA interference by the RISC complex.
Project description:RNA-mediated gene silencing in human cells requires the accurate generation of ?22 nt microRNAs (miRNAs) from double-stranded RNA substrates by the endonuclease Dicer. Although the phylogenetically conserved RNA-binding proteins TRBP and PACT are known to contribute to this process, their mode of Dicer binding and their genome-wide effects on miRNA processing have not been determined. We solved the crystal structure of the human Dicer-TRBP interface, revealing the structural basis of the interaction. Interface residues conserved between TRBP and PACT show that the proteins bind to Dicer in a similar manner and by mutual exclusion. Based on the structure, a catalytically active Dicer that cannot bind TRBP or PACT was designed and introduced into Dicer-deficient mammalian cells, revealing selective defects in guide strand selection. These results demonstrate the role of Dicer-associated RNA binding proteins in maintenance of gene silencing fidelity.
Project description:The cytoplasmic RNA-induced silencing complex (RISC) contains dsRNA binding proteins, including protein kinase RNA activator (PACT), transactivation response RNA binding protein (TRBP), and Dicer, that process pre-microRNAs into mature microRNAs (miRNAs) that target specific mRNA species for regulation. There is increasing evidence for important functional interactions between the miRNA and nuclear receptor (NR) signaling networks, with recent data showing that estrogen, acting through the estrogen receptor, can modulate initial aspects of nuclear miRNA processing. Here, we show that the cytoplasmic RISC proteins PACT, TRBP, and Dicer are steroid receptor RNA activator (SRA) binding NR coregulators that target steroid-responsive promoters and regulate NR activity and downstream gene expression. Furthermore, each of the RISC proteins, together with Argonaute 2, associates with SRA and specific pre-microRNAs in both the nucleus and cytoplasm, providing evidence for links between NR-mediated transcription and some of the factors involved in miRNA processing.