Publications by authors named "Gatikrushna Singh"

15 Publications

  • Page 1 of 1

A New Approach to 3D Modeling of Inhomogeneous Populations of Viral Regulatory RNA.

Viruses 2020 09 29;12(10). Epub 2020 Sep 29.

Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA.

Tertiary structure (3D) is the physical context of RNA regulatory activity. Retroviruses are RNA viruses that replicate through the proviral DNA intermediate transcribed by hosts. Proviral transcripts form inhomogeneous populations due to variable structural ensembles of overlapping regulatory RNA motifs in the 5'-untranslated region (UTR), which drive RNAs to be spliced or translated, and/or dimerized and packaged into virions. Genetic studies and structural techniques have provided fundamental input constraints to begin predicting HIV 3D conformations in silico. Using SimRNA and sets of experimentally-determined input constraints of HIV trans-activation responsive sequence (TAR) and pairings of unique-5' (U5) with dimerization (DIS) or AUG motifs, we calculated a series of 3D models that differ in proximity of 5'-Cap and the junction of TAR and PolyA helices; configuration of primer binding site (PBS)-segment; and two host cofactors binding sites. Input constraints on U5-AUG pairings were most compatible with intramolecular folding of 5'-UTR motifs in energetic minima. Introducing theoretical constraints predicted metastable PolyA region drives orientation of 5'-Cap with TAR, U5 and PBS-segment helices. SimRNA and the workflow developed herein provides viable options to predict 3D conformations of inhomogeneous populations of large RNAs that have been intractable to conventional ensemble methods.
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http://dx.doi.org/10.3390/v12101108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650772PMC
September 2020

The mRNA encoding the JUND tumor suppressor detains nuclear RNA-binding proteins to assemble polysomes that are unaffected by mTOR.

J Biol Chem 2020 05 20;295(22):7763-7773. Epub 2020 Apr 20.

Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, Minnesota 55108

One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT-quantitative PCR results revealed that mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the -containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of -containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether -NCBP1/NCBP3 to polysomes. We also found that translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.
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http://dx.doi.org/10.1074/jbc.RA119.012005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261793PMC
May 2020

The Long Noncoding RNA Regulates HIV-1 Replication through Epigenetic Regulation of the HIV-1 Promoter.

mBio 2019 09 24;10(5). Epub 2019 Sep 24.

Division of Genetics, Department of Pediatrics, UCSD Center for AIDS Research, and Institute for Genomic Medicine, University of California San Diego, La Jolla, California, USA

A major challenge in finding a cure for HIV-1/AIDS is the difficulty in identifying and eradicating persistent reservoirs of replication-competent provirus. Long noncoding RNAs (lncRNAs, >200 nucleotides) are increasingly recognized to play important roles in pathophysiology. Here, we report the first genome-wide expression analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs). We identified an lncRNA, which we named IV-1-nhnced ncRNA (), that is upregulated by HIV-1 infection of MDMs, microglia, and T lymphocytes. Peripheral blood mononuclear cells of HIV-1-infected individuals show elevated levels of Importantly, is a broad enhancer of multiple HIV-1 strains because depletion of inhibited X4, R5, and dual-tropic HIV replications and the inhibition was rescued by overexpression. forms a complex with the RNA-binding protein FUS, which facilitates HIV replication through at least two mechanisms: (i) -FUS complex binds the HIV promoter and enhances recruitment of the histone acetyltransferase p300, which positively regulates HIV transcription by increasing histone H3K27 acetylation and P-TEFb enrichment on the HIV promoter, and (ii) -FUS complex is enriched at the promoter of the cyclin-dependent kinase 2 gene, , to enhance CDK2 expression. Notably, knockdown and knockout mediated by RNA interference (RNAi) and CRISPR-Cas9, respectively, prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment Our results suggest that silencing of or perturbation of the -FUS ribonucleoprotein complex could provide a new epigenetic silencing strategy to eradicate viral reservoirs and effect a cure for HIV-1/AIDS. Despite our increased understanding of the functions of lncRNAs, their potential to develop HIV/AIDS cure strategies remains unexplored. A genome-wide analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs) was performed, and 1,145 differentially expressed lncRNAs were identified. An lncRNA named IV-1-nhnced ncRNA () is upregulated by HIV-1 infection and promotes HIV replication in T cells and macrophages. forms a complex with the RNA-binding protein FUS to enhance transcriptional coactivator p300 recruitment to the HIV promoter. Furthermore, knockdown and knockout prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment, suggesting as a potential therapeutic target to cure HIV-1/AIDS.
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http://dx.doi.org/10.1128/mBio.02016-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759764PMC
September 2019

Virion-associated, host-derived DHX9/RNA helicase A enhances the processivity of HIV-1 reverse transcriptase on genomic RNA.

J Biol Chem 2019 07 7;294(30):11473-11485. Epub 2019 Jun 7.

Department of Biochemistry, University of Missouri, Columbia, Missouri 65211

DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNA-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.
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http://dx.doi.org/10.1074/jbc.RA119.007679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6663884PMC
July 2019

HIV-1 Escape from Small-Molecule Antagonism of Vif.

mBio 2019 02 26;10(1). Epub 2019 Feb 26.

Department of Medicine, University of Miami Miller School of Medicine, Miami, Florida, USA

The HIV-1 accessory protein Vif, which counteracts the antiviral action of the DNA-editing cytidine deaminase APOBEC3G (A3G), is an attractive and yet unexploited therapeutic target. Vif reduces the virion incorporation of A3G by targeting the restriction factor for proteasomal degradation in the virus-producing cell. Compounds that inhibit Vif-mediated degradation of A3G in cells targeted by HIV-1 would represent a novel antiviral therapeutic. We previously described small molecules with activity consistent with Vif antagonism. In this study, we derived inhibitor escape HIV-1 variants to characterize the mechanism by which these novel agents inhibit virus replication. Here we show that resistance to these agents is dependent on an amino acid substitution in Vif (V142I) and on a point mutation that likely upregulates transcription by modifying the lymphocyte enhancing factor 1 (LEF-1) binding site. Molecular modeling demonstrated a docking site in the Vif-Elongin C complex that is disrupted by these inhibitors. This docking site is lost when Vif acquires the V142I mutation that leads to inhibitor resistance. Competitive fitness experiments indicated that the V142I Vif and LEF-1 binding site mutations created a virus that is better adapted to growing in the presence of A3G than the wild-type virus. Although antiretroviral therapy can suppress HIV-1 replication effectively, virus reservoirs persist in infected individuals and virus replication rapidly rebounds if therapy is interrupted. Currently, there is a need for therapeutic approaches that eliminate, reduce, or control persistent viral reservoirs if a cure is to be realized. This work focuses on the preclinical development of novel, small-molecule inhibitors of the HIV-1 Vif protein. Vif inhibitors represent a new class of antiretroviral drugs that may expand treatment options to more effectively suppress virus replication or to drive HIV-1 reservoirs to a nonfunctional state by harnessing the activity of the DNA-editing cytidine deaminase A3G, a potent, intrinsic restriction factor expressed in macrophage and CD4 T cells. In this study, we derived inhibitor escape variants to characterize the mechanism by which these novel agents inhibit virus replication and to provide evidence for target validation.
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http://dx.doi.org/10.1128/mBio.00144-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6391917PMC
February 2019

Identification of conserved, primary sequence motifs that direct retrovirus RNA fate.

Nucleic Acids Res 2018 08;46(14):7366-7378

Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN 55108, USA.

Precise stoichiometry of genome-length transcripts and alternatively spliced mRNAs is a hallmark of retroviruses. We discovered short, guanosine and adenosine sequence motifs in the 5'untranslated region of several retroviruses and ascertained the reasons for their conservation using a representative lentivirus and genetically simpler retrovirus. We conducted site-directed mutagenesis of the GA-motifs in HIV molecular clones and observed steep replication delays in T-cells. Quantitative RNA analyses demonstrate the GA-motifs are necessary to retain unspliced viral transcripts from alternative splicing. Mutagenesis of the GA-motifs in a C-type retrovirus validate the similar downregulation of unspliced transcripts and virion structural protein. The evidence from cell-based co-precipitation studies shows the GA-motifs in the 5'untranslated region confer binding by SFPQ/PSF, a protein co-regulated with T-cell activation. Diminished SFPQ/PSF or mutation of either GA-motif attenuates the replication of HIV. The interaction of SFPQ/PSF with both GA-motifs is crucial for maintaining the stoichiometry of the viral transcripts and does not affect packaging of HIV RNA. Our results demonstrate the conserved GA-motifs direct the fate of retrovirus RNA. These findings have exposed an RNA-based molecular target to attenuate retrovirus replication.
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http://dx.doi.org/10.1093/nar/gky369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6101577PMC
August 2018

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution.

J Vis Exp 2017 01 16(119). Epub 2017 Jan 16.

Department of Veterinary & Biomedical Sciences, University of Minnesota; Department of Veterinary Biosciences, Ohio State University;

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes. The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.
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http://dx.doi.org/10.3791/54391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5352254PMC
January 2017

RNAi Screening in Spodoptera frugiperda.

Methods Mol Biol 2016 ;1470:199-212

Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India.

RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line.
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http://dx.doi.org/10.1007/978-1-4939-6337-9_16DOI Listing
December 2017

1,2,3-Triazoles as Amide Bioisosteres: Discovery of a New Class of Potent HIV-1 Vif Antagonists.

J Med Chem 2016 08 10;59(16):7677-82. Epub 2016 Aug 10.

Program for RNA Biology, Sanford Burnham Prebys Medical Discovery Institute , La Jolla, California 92037, United States.

RN-18 based viral infectivity factor (Vif), Vif antagonists reduce viral infectivity by rescuing APOBEC3G (A3G) expression and enhancing A3G-dependent Vif degradation. Replacement of amide functionality in RN-18 (IC50 = 6 μM) by isosteric heterocycles resulted in the discovery of a 1,2,3-trizole, 1d (IC50 = 1.2 μM). We identified several potent HIV-1 inhibitors from a 1d based library including 5ax (IC50 = 0.01 μM), 5bx (0.2 μM), 2ey (0.4 μM), 5ey (0.6 μM), and 6bx (0.2 μM).
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http://dx.doi.org/10.1021/acs.jmedchem.6b00247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5534211PMC
August 2016

Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

Methods Mol Biol 2016 ;1354:133-46

Department of Veterinary Biosciences, The Ohio State University, 1900 Coffey Road, Columbus, OH, USA.

All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.
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http://dx.doi.org/10.1007/978-1-4939-3046-3_9DOI Listing
October 2016

HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.

Virology 2015 Dec 13;486:307-20. Epub 2015 Nov 13.

Department Veterinary & Biomedical Sciences, University of Minnesota, 205 VSB, 1971 Commonwealth Avenue, Saint Paul, MN 55108. Electronic address:

Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.
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http://dx.doi.org/10.1016/j.virol.2015.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877169PMC
December 2015

SAR and Lead Optimization of an HIV-1 Vif-APOBEC3G Axis Inhibitor.

ACS Med Chem Lett 2012 Jun;3(6):465-469

Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla, California 92037 (USA).

We describe structure-activity relationship and optimization studies of RN-18, an HIV-1 Vif-APOBEC3G axis inhibitor. Targeted modifications of RN-18 ring-C, ring-B, ring-A, bridge A-B, and bridge B-C were performed to identify the crucial structural features, which generated new inhibitors with similar ( and ) and improved (, , and ) activities. Two potent water-soluble RN-18 analogues, and are also disclosed, and we describe the results of pharmacological studies with compound The findings described here will be useful in the development of more potent Vif inhibitors and in the design of probes to identify the target protein of RN-18 and its analogues.
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http://dx.doi.org/10.1021/ml300037kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3922209PMC
June 2012

Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda.

Appl Environ Microbiol 2010 Nov 10;76(21):7202-9. Epub 2010 Sep 10.

Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.
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http://dx.doi.org/10.1128/AEM.01552-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2976263PMC
November 2010

Systematic deletion and site-directed mutagenesis of FHVB2 establish the role of C-terminal amino acid residues in RNAi suppression.

Biochem Biophys Res Commun 2010 Jul 25;398(2):290-5. Epub 2010 Jun 25.

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.

Viruses and siRNA/miRNA machinery of the host cell interact in diverse ways with the virus encoded RNAi suppressor proteins. These interactions have implications on the replication and pathogenicity of the virus and also on the immune response of the host. Suppressor protein B2 of insect Flock House Virus (FHVB2), has been shown to mediate RNAi suppression via N-terminal region by directly binding to dsRNA. We have previously shown that FHVB2 protein also interacts with host Dicer protein via its PAZ domain. In the present study, we performed systematic mutagenesis studies to map the FHVB2 regions involved in mediating suppression of RNAi. Progressive deletion of 17 amino acids from N- and C-terminii of FHVB2 resulted in cumulative decrease in RNAi suppression activity of FHVB2. The deletion of 17 amino acids from the C-terminus resulted in more reduction in RNAi suppression in comparison to the N-terminal deletions. Subsequently, we generated 17 successive point mutants of FHVB2 C-terminus and evaluated the RNAi suppression activity for each of the point mutants. Each point mutation resulted in a significant reduction in RNAi suppression activity of FHVB2. These results provide evidence for the role of C-terminal of FHVB2 in RNAi suppression.
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http://dx.doi.org/10.1016/j.bbrc.2010.06.083DOI Listing
July 2010

Suppression of RNA silencing by Flock house virus B2 protein is mediated through its interaction with the PAZ domain of Dicer.

FASEB J 2009 Jun 4;23(6):1845-57. Epub 2009 Feb 4.

International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India.

RNA silencing is a conserved pathway that functions as an antiviral mechanism. The majority of viruses encode silencing suppressors that interfere with siRNA- and miRNA-guided silencing pathways. The insect flock house virus B2 protein (FHVB2) functions as an RNAi silencing suppressor that inhibits siRNA biogenesis. Here, we describe the generation of a GFP silent sensor line (Sf21) and a GFP sensor line expressing FHVB2 to study RNAi suppression mechanisms. Overexpression of FHVB2 resulted in suppression of GFP-RNAi and resumption of GFP expression. Protein fractionation studies with FHVB2-transfected cells showed that FHVB2 associates with a high-molecular-weight complex of Dicer and dsRNA/siRNAs. Yeast two-hybrid and pulldown assays revealed an interaction between FHVB2 and Drosophila Dicer proteins that appeared to involve PAZ domains. To map the FHVB2 domains interacting with Dicer, we used a 17-residue C-terminal deletion mutant. RNAi suppression was reversed in cells transfected with the FHVB2 mutant as revealed by loss of GFP. Additional yeast two-hybrid and in vitro pulldown assays confirmed that the C-terminal region of FHVB2 was involved in the interaction with the PAZ domains of Dicers. These results thus reveal a novel interaction between FHVB2 and Dicer that leads to suppression of siRNA biogenesis.
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http://dx.doi.org/10.1096/fj.08-125120DOI Listing
June 2009