Molecular determinants of cytochrome C oxidase IV mRNA axonal trafficking.
In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.
Section on Neurobiology, Laboratory of Molecular Biology, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA.
Amar N
AN
Kar
Section on Neurobiology, Laboratory of Molecular Biology, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA.
Jose Norberto S
JNS
Vargas
Section on Neurobiology, Laboratory of Molecular Biology, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA.
Cai-Yun
CY
Chen
NIMH-NINDS Clinical Proteomics Unit, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA.
Jeffrey A
JA
Kowalak
Section on Neurobiology, Laboratory of Molecular Biology, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA.
Anthony E
AE
Gioio
Section on Neurobiology, Laboratory of Molecular Biology, Division of Intramural Research Programs, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA. Electronic address: barry.kaplan@nih.gov.
Barry B
BB
Kaplan
D000818:Animals; D000831:Animals, Newborn; D001369:Axons / Q000378:metabolism*; D002478:Cells, Cultured; D003576:Electron Transport Complex IV / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D034702:RNA-Binding Protein FUS / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017783:Superior Cervical Ganglion / Q000166:cytology*; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology; D051840:Y-Box-Binding Protein 1 / Q000235:genetics / Q000378:metabolism
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034702:RNA-Binding Protein FUS; D016601:RNA-Binding Proteins; D051840:Y-Box-Binding Protein 1; D014212:Tretinoin; D003576:Electron Transport Complex IV; C573472:ATP synthase subunit 9, rat; D025261:Mitochondrial Proton-Translocating ATPases
FUS; RNA binding proteins; Sympathetic neurons; YB-1; mRNA localization; mRNA trafficking