JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_4.rawhttps://storage.jpostdb.org/JPST003462/files/230301izumi_230220-NCL2_3.rawhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_1.rawhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_3.rawhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_5.rawhttps://storage.jpostdb.org/JPST003462/files/230301izumi_230220-NCL2_2.rawhttps://storage.jpostdb.org/JPST003462/files/230301izumi_230220-NCL2_1.rawhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_2.rawhttps://storage.jpostdb.org/JPST003462/files/230301izumi_230220-NCL2_2.raw.mgfhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_3.raw.mgf-123444.xlsxhttps://storage.jpostdb.org/JPST003462/files/230301izumi_230220-NCL2_2.raw.mgf-125944.xlsxhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_2.raw.mgfhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_3.raw.mgfhttps://storage.jpostdb.org/JPST003462/files/230301izumi_230220-NCL2_1.raw.mgf-125558.xlsxhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_4.raw.mgfhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_2.raw.mgf-123214.xlsxhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_1.raw.mgf-123030.xlsxhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_4.raw.mgf-123750.xlsxhttps://storage.jpostdb.org/JPST003462/files/230512_Rcpt20230324_izumi_1.raw.mgfhttps://storage.jpostdb.org/JPST003462/files/230301izumi_230220-NCL2_1.raw.mgfprimaryOK200ProteomicsMasami NagahamaHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST003462Meiji pharmaceutical universityjPOSTWD repeat domain 74 (WDR74) is a nucleolar protein involved in the early stages of pre-60S maturation in the ribosome biogenesis pathway. In later stages, WDR74 interacts with MTR4, an RNA helicase that functions with the exosome nuclease complex, and is dissociated upon ATP hydrolysis by the chaperone-like nuclear VCP-like 2 (NVL2) AAA-ATPase. We previously reported that ATP hydrolysis-defective NVL2 causes aberrant accumulation of WDR74 on the MTR4-exosome complex at the nucleolar periphery and in the nucleoplasm and that this nuclear redistribution of WDR74 leads to the unusual cleavage of the early rRNA precursor within the internal transcribed spacer 1 sequence. However, the precise mechanisms underlying this NVL2-mediated regulation is largely obscure. In this study, co-immunoprecipitation combined with mass spectrometry revealed that WDR74 functions as part of a pre-ribosomal subcomplex, termed the WDR74 module, consisting of eukaryotic conserved WDR74, RPF1, MAK16, and RRP1. Each component of the WDR74 module was mutually essential for the interaction of other members with MTR4, and all components were required for the accurate cleavage of pre-rRNA during 60S ribosome biogenesis. Moreover, impaired release of WDR74 from the MTR4-exosome complex caused by NVL2 dysfunction prevented MTR4 from recruiting PICT1, an MTR4 adaptor protein required for the 3'-end maturation of 5.8S rRNA. Our results highlight the key role of the WDR74 module in coordinating the early pre-rRNA cleavage and late processing of pre-5.8S rRNAs.Pre-ribosomal WDR74 module coordinates the early and late pre-rRNA processing stages for the NVL2-mediated regulation of 60S ribosome biogenesis.Hirooka Yuya Y, Izumikawa Keiichi K, Miyao Sotaro S, Ohga Takayuki T, Nobe Yuko Y, Taoka Masato M, Nagahama Masami MABETA, Co-Immunoprecipitations, Dnahc8, RRP1, PhrB photolyase activity, Repeat, snRNA Precursor, ALADIN, 4-d][1, RGD1311297, RNA Transcript, ADRACALA, cleavage, Ter94, CTFgamma, protein, pigmented epithelium, ALS14, Social Controls, ter, Co Immunoprecipitation, WD40, AAA, DmelCG2331, extracellular vesicular exosome complex, WDR74, Roles, Nucleolar Protein, Mtr4, DNA-Dependent ATPase, pathogenesis, Concepts, extracellular vesicular exosome, 3, NUP96, Analysis, Transfer, protein aggregate, Fs(3)Hor, epithelium, DmTER94, Formal Social Controls, ATPases, IKKg, KEY, Key, Mass Spectrum Analysis, Ribosomal, DmelCG2684, Nuclear Protein, Pre mRNA, TERA, I, Analyses, achalasia alacrima syndrome, BXDC5, Addisonian achalasia syndrome, AAAS, Rbm13, Pre-snRNA, Co-Immunoprecipitation, 15S Mg(2+)-ATPase p97 subunit, dvcp, pigmented retina, Precipitation, 2310066N05Rik, NTef2, p97/VCP, NNP-1, DNA cyclobutane dipyrimidine photolyase activity, RBM13, SUPPRESSOR OF AUXIN RESISTANCE 3, WD40 Repeat, achalasia-alacrima syndrome, Social, Transcript, 5730436H21Rik, IBMPFD, ATP hydrolysis, Pre rRNA, Immune, l(2)03775, Adenosinetriphosphatase, DNA-Dependent Adenosinetriphosphatases, Repeats, 22.26, 4-d) 1, ter94, AD1, Role Concepts, dVCP, 2600016B03Rik, APPI, Pre-tRNA, Precipitations, WD Repeats, F23A5_3., PRE, alacrima-achalasia-adrenal insufficiency neurologic disorder, aberrant, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, Hst6.7b, KIAA0179, CG2331, Spectrum Analysis, Fs(3)Sz11, Spectroscopy, 3.6.4.6, dmTERA, tRNA, NNP1L, DmIKKgamma, RNA HELICASE, Tera, Role Concept, retinal pigment, ADRACALIN, dIKK, KIAA0052, Kenny, Role, dipyrimidine photolyase (photosensitive), BcDNA:GM02885, DNA-Dependent, ATP phosphohydrolase, RNA Primary Transcript, Addisonian-achalasia syndrome, Bxdc5, END, retinal pigment layer, Valosin-containing protein, exosome complex, NEDD4-1, WD, Pre snRNA, 3]dioxole-5-carboxylic acid, HEL-220, IKK-gamma, Control, Spectrometry, Triphosphatase, 4-methylenedioxy-8-methoxy-10-nitro-1-phenanthrenecarboxylic acid, anon-WO2004063362.65, Controls, anon-WO2004063362.67, early, Adenosinetriphosphatases, AA407588, Adenosine, phr A photolyase activity, DNA-photoreactivating enzyme, DmelCG16910, VCP/p97, Pre-mRNA, RRP1A, Horka, p97|VCP, achalasia addisonianism alacrimia syndrome, CG2684, Fs(3)Horka, Apple, TER ATPase, ACTH-resistant adrenal insufficiency, Regulation, exosome, Regulations, HEL-S-70, atypia, Gene, Precursor RNA, Spectrum Analyses, photoreactivating enzyme activity, protein-containing complex, dIKK-gamma, P1-Loop, 7.12, tRNA Precursor, beta-Transducin, GL003, D21S2056E, Messenger, MAK16L, Helicases, DmIKK-gamma, Pre-rRNA, Immune Precipitations, Mass, Gene Products, Primary RNA Transcript, RP11-118B23.1, stratum pigmentosa retinae, DmF2, atypical, alacrima-achalasia-addisonianism, dmIKKgamma, VCP ATPase, glucocorticoid deficiency and achalasia, Pre tRNA, IKK[[gamma]], aristolochic acid-I, Immune Precipitation, Mass Spectroscopy, lod, VCP, Vcp, study, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, PRECOCIOUS, AI314911, RPF1, dysfunction, F23A5.3, causes, DmelCG7845, WD40 Motifs, AU018604, ATPase, 8-methoxy-6-nitrophenanthol (3, IKK, rbm13, dTERA, causality, Motifs, DNA Dependent, TAA, beta Transducin Repeats, F23A5_3, rpf1, HHT1, AL033306, DNA Dependent ATPase, RNA, cdc48, Nnp1, AL033305, Edg, Aristolochic acid, Formal Social Control, protein complex, Precursor, p97, PPP1R136, Proteins, mak16, Precursors, defective, Primary, IBMPFD1, Concept, Aristolochic acid I, DNA Dependent Adenosinetriphosphatases, rRNA Precursor, IKKgamma, MODIFIER OF SNC1, MS, MMI9_2, native protein, achalasia and alacrima, Social Control, mRNA Precursor, Protein, WD40 Motif, deoxyribonucleic photolyase activity, MMI9.2, sequence, triple A syndrome, pTER94, Nuclear, Small Nuclear, mak16l, BcDNA.GM02885, 2210420E24Rik, Mass Spectrum Analyses, 3-dioxole-5-carboxylic acid, 8-methoxy-6-nitrophenanthro[3, Mass Spectrum, PN-II, RPE, Adenosine Triphosphatase, hdhc9, mRNA, photolyase activity, Dmikkgamma, snRNA, PICT1, RNA Precursor, 3110001E05, aristolochin, Nucleolar Proteins, CDC48, pathophysiology, Lds, Dob1, DOB1, RNA Primary, CG16910, Primary RNA, primary structure of sequence macromolecule, l(2)46Ch, p. pigmentosa retinae, Protein Gene Products, Helicase, beta-Transducin Repeat, AAASb, Gene Proteins, WD Repeat, rRNA, PICT-1, l(2)46CFs, fSAP118, beta-Transducin Repeats, RNA Helicase, ribosome biogenesis and assembly, deoxyribonucleate pyrimidine dimer lyase (photosensitive), regulation, PN2, Primary Transcript, ORW1, Nucleolar, l(2)46CFf, D030041N15Rik, NOP52, CVAP, hypoadrenalism with achalasia, Motif, ABPPVCP, HEL-S-70, Vcp, cdc48, TERA, I, Valosin-containing protein., p97, 15S Mg(2+)-ATPase p97 subunit, dvcp, HEL-220, Ter94, 3110001E05, CDC48, CG2331, anon-WO2004063362.65, p97/VCP, anon-WO2004063362.67, ALS14, IBMPFD1, l(2)46Ch, ter, 7.12, 3.6.4.6, IBMPFD, DmelCG2331, dmTERA, l(2)03775, Tera, VCP/p97, l(2)46CFs, 22.26, p97|VCP, dTERA, ter94, dVCP, Apple, BcDNA:GM02885, pTER94, TER ATPase, VCP ATPase, BcDNA.GM02885, DmTER94, l(2)46CFfliquid chromatography tandem mass spectroscopy, LC-MS2, PRE, Cognitive Function, MGC130048, SGCG_HUMAN, PhrB photolyase activity, determination, 35 kDa dystrophin-associated glycoprotein, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, Proteins, LC-MS/MS, SG-gamma, A4, Gene, photoreactivating enzyme activity, Insight, pigmented epithelium, TYPE, SGCG, LC-MS-MS, LGMD2C, DAGA4, Insights, LC/MS/MS, MODIFIER OF SNC1, retinal pigment, LC-MSMS, chemical analysis, Protein, deoxyribonucleic photolyase activity, 35DAG, Gene Products, dipyrimidine photolyase (photosensitive), 3, sarcoglycan, stratum pigmentosa retinae, NUP96, MAM, gamma-SG, epithelium, SCG3, retinal pigment layer, gamma sarcoglycan, MOS3, RPE, LCMSMS, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, DMDA1, PRECOCIOUS, photolyase activity, Function, pigmented retina, F23A5.3, Cognitive, gamma (35kDa dystrophin-associated glycoprotein), DNA cyclobutane dipyrimidine photolyase activity, SUPPRESSOR OF AUXIN RESISTANCE 3, p. pigmentosa retinae, Cognitive Functions, Protein Gene Products, Gene Proteins, phr A photolyase activity, Cognitions, DMDA, DNA-photoreactivating enzyme, Functions, liquid chromatography-tandem mass spectroscopy, 35kD dystrophin-associated glycoprotein, SCARMD2, gamma-sarcoglycan, liquid chromatography tandem mass spectrometry, LC-MS-MS., deoxyribonucleate pyrimidine dimer lyase (photosensitive), assay, F23A5_3Regulations, PRE, RNA, PhrB photolyase activity, snRNA Precursor, Formal Social Control, Precursor, RNA Transcript, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, Precursors, Precursor RNA, Primary, photoreactivating enzyme activity, pigmented epithelium, Social Controls, Fs(3)Sz11, tRNA Precursor, rRNA Precursor, tRNA, MODIFIER OF SNC1, WDR74, retinal pigment, Social Control, Messenger, mRNA Precursor, deoxyribonucleic photolyase activity, Pre-rRNA, dipyrimidine photolyase (photosensitive), Primary RNA Transcript, 3, stratum pigmentosa retinae, Small Nuclear, NUP96, DmF2, Transfer, Pre tRNA, RNA Primary Transcript, Fs(3)Hor, epithelium, retinal pigment layer, Formal Social Controls, lod, Ribosomal, DmelCG2684, MOS3, RPE, Pre mRNA, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, PRECOCIOUS, mRNA, photolyase activity, Pre snRNA, snRNA, Pre-snRNA, pigmented retina, RNA Precursor, Control, F23A5.3, ribosome biogenesis and assembly., NTef2, Controls, Lds, RNA Primary, Primary RNA, DNA cyclobutane dipyrimidine photolyase activity, early, SUPPRESSOR OF AUXIN RESISTANCE 3, p. pigmentosa retinae, Social, AA407588, Transcript, DmelCG7845, 5730436H21Rik, phr A photolyase activity, Pre rRNA, rRNA, DNA-photoreactivating enzyme, Pre-mRNA, Horka, CG2684, Fs(3)Horka, deoxyribonucleate pyrimidine dimer lyase (photosensitive), regulation, Pre-tRNA, Primary Transcript, Regulation, F23A5_3falseNuclear VCP-like 2 (NVL2)-associated complexLC-MSMS analysis of NVL2-associated pre-60S complex. To obtain more insights into the molecular mechanism of this effects caused by the mutant NVL2 in pre-60S complexes,we searched for additional proteins that preferentially interact with the NVL2(E365Q/E682Q) in comparison with wild-type NVL2. Co-mmunoprecipitation was performed by using FLAG-tagged NVL2 (wild-type or E365Q/E682Q) as baits and their interacting proteins were analyzed by LC-MS/MS.Fri Jan 31 00:00:00 GMT 2025PXD057741960639706051