JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST001051/files/170523mura01_170523taDS_0p2ul_uBM17_pSTY_PSM.xlsxhttps://storage.jpostdb.org/JPST001051/files/170523mura02_170523taDS_0p2ul_uBM17_pSTY_PSM.xlsxhttps://storage.jpostdb.org/JPST001051/files/170523mura01_170523taDS_0p2ul.rawhttps://storage.jpostdb.org/JPST001051/files/170523mura02_170523taDS_0p2ul.rawhttps://storage.jpostdb.org/JPST001051/files/170523mura01_170523taDS_0p2ul.mzXMLhttps://storage.jpostdb.org/JPST001051/files/170523mura02_170523taDS_0p2ul.mzXMLprimaryOK200ProteomicsMikiko C. SiomiBombyx Morihttps://repository.jpostdb.org/entry/JPST001051The University of TokyojPOSTThe piRNA amplification pathway in Bombyx is operated by Ago3 and Siwi in their piRISC form. The DEAD-box protein, Vasa, facilitates Ago3-piRISC production by liberating cleaved RNAs from Siwi-piRISC in an ATP hydrolysis-dependent manner. However, the Vasa-like factor facilitating Siwi-piRISC production along this pathway remains unknown. Here, we identify DEAD-box polypeptide 43 (DDX43) as the Vasa-like protein functioning in Siwi-piRISC production. DDX43 belongs to the helicase superfamily II along with Vasa, and it contains a similar helicase core. DDX43 also contains a K-homology (KH) domain, a prevalent RNA-binding domain, within its N-terminal region. Biochemical analyses show that the helicase core is responsible for Ago3-piRISC interaction and ATP hydrolysis, while the KH domain enhances the ATPase activity of the helicase core. This enhancement is independent of the RNA-binding activity of the KH domain. For maximal DDX43 RNA-binding activity, both the KH domain and helicase core are required. This study not only provides new insight into the piRNA amplification mechanism but also reveals unique collaborations between the two domains supporting DDX43 function within the pathway.DEAD-box polypeptide 43 facilitates piRNA amplification by actively liberating RNA from Ago3-piRISC.Murakami Ryo R, Sumiyoshi Tetsutaro T, Negishi Lumi L, Siomi Mikiko C MCProtein Gene Products, Gene Proteins, EIF2C3, DmelCG40300, BEST:LD15785, ago3, C130014L07Rik, Ago-3, Protein., dAgo3, Protein, CG40300, Gene Products, Proteins, AW048688, Gene, Ago3, Eif2c3pf7, Forms, deceased, CG33678, Ribonucleic, Vas, VAS, Activity, Bombyx mori, polypeptide sequence, DmRH25, vasa, VASA, DmelCG4919, adenosinetriphosphatase activity, Gene, Si-11, protein, Cardiac Death, CG3506, protein-containing complex, peptide, Polypeptides, BEST:LD15785, RP11-539G11.1, ATP hydrolysis-dependent activity, ATP monophosphatase activity, Gene Products, Eif2c3, DmelCG43081, protein aggregate, CG43081, Non Polyadenylated, Aggravating interaction, RNA Gene Products, adenosine triphosphatase activity, Silkmoths, study, EIF2C3, (Ca2+ + Mg2+)-ATPase, AV206478, complex V (mitochondrial electron transport), dead, CT13, ligand, polypeptides, DmGCSl, ATPase activity, Silkmoth, GCLm, Non-Polyadenylated RNA, BG:DS00929.15, ago3, BG:DS00929.14, mori, Dmel_CG33678, ENSMUSG00000070291, dAgo3, Silkworms, Vasa, AW048688, site, Gcsl, GCSl, Ago3, 4919, CG4919, RNA, ATP hydrolase activity, Ago-3, polipeptido, ribose nucleic acid, protein complex, Bombyx, EP(2)0812, adenosine 5'-triphosphatase activity, Proteins, fs(2)ltoRJ36, cgt, ribonucleic acids, Dmel_CG3506, RNS, Peptide, MER3, polypeptide, Si-11-6, native protein, ATPase-dependent activity, <new synonym>, DmGCLM, OTTMUSG00000019690, yeast nucleic acid, Ribonukleinsaeure, Protein, HCO3--ATPase, post-mortem, core, sequence, pentosenucleic acids, Ribonucleic acids, HAGE, SEC63D1, Bombyx morus, Death, region, coupled, Mvh, ATP phosphohydrolase activity, Bombyxs, ribonucleic acid, 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F10B6_15, rac-Dithiothreitol, Dinitrilotetraacetate, Endopeptidase Inhibitor, Ethylenedinitrilotetraacetic Acid, threo-1, Endopeptidase Inhibitors, Reagent, Cleland's reagent, Potassium EDTA, Ethylenediaminetetraacetic, Peptide Hydrolase Inhibitor, 4-dimercapto-, HEPES Monosodium Salt, Protease, Ethylenedinitrilotetraacetic, SDH, B-cell receptor complex, R*)-, Calcium Disodium, F10B6.15, Buffer, Dicobalt, Co Immunoprecipitation, Potassium Salt, antibody, SDS, BEST:LD15785, BCR complex, Edetic, Magnesium Disodium, Edetic Acid, congenital, Protease Inhibitor, Immune Precipitations, 1, 2, Inhibitors, Protease Antagonist, Eif2c3, Peptide Hydrolase, Hydrolase Inhibitors, immunoglobulin complex, Immune Precipitation, Calcium Tetacine, (R*, Disodium Salt, HEPES Monosodium, EIF2C3, Edetates, Ethylene, Glycine, membrane bound, N, Co-Immunoprecipitation, Monosodium Salt, Salt, Precipitation, 4733401P19Rik, Antagonist, Edetate, free, Calcitetracemate, lipomatosis of pancreas, Calcium Disodium 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Ethylenediaminetetraacetic AcidfalseAgo3 interaction proteins in BmN4 cellWe prepared BmN4 cell lysates in Co-IP buffer [30 mM HEPES-KOH pH 7.3, 150 mM potassium acetate, 5 mM magnesium acetate, 5 mM dithiothreitol, 0.1% (w/v) NP-40 and cOmplete ULTRA EDTA-free protease inhibitor (Roche)] with and without 1 mM EDTA and conducted immunoprecipitation assays using anti-Ago3 antibody. The reaction was terminated by boiling with SDS sample buffer.Fri Jan 08 00:00:00 GMT 2021PXD023467709133555135