Seminar: Dr. Barry Kaplan

Molecular determinants of axonal mRNA trafficking

Date:
16 June 2015 15:30 hrs. - 16:30 hrs.
Location:
Figdor Lecture Theatre, 8th floor RIMLS Building, Geert Grooteplein 26-28, route 289
Title:
Molecular determinants of axonal mRNA trafficking
Speaker(s):

Dr. Barry Kaplan, National Institute of Mental Health, Bethesda, USA

Host(s):

Prof. Gerard Martens, Dept. of Molecular Animal Physiology. theme  Neurodevelopmental disorders

16-06-2015 15:30:0016-06-2015 16:30:00Europe/AmsterdamMolecular determinants of axonal mRNA trafficking Figdor Lecture Theatre, 8th floor RIMLS Building, Geert Grooteplein 26-28, route 289Rimlsrimls@radboudumc.nl

Remarks / more information:

undefinedIn previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3’-untranslated region of the COXIV transcript that was necessary and sufficient for the axonal localization of COXIV mRNA 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 transcript.  

To identify proteins involved in the axonal transport of the COXIV mRNA, we used this 38-nucleotide COXIV RNA zip-code as bait for RNA protein binding studies. Gel-shift assays using the biotinylated COXIV-zipcode indicated that the zipcode binds proteins and forms ribonucleoprotein complexes. To identify proteins present in the COXIV-zipcode ribonucleoprotein complex we performed a mass spectrometric (MS) analysis of the biotinylated zipcode affinity purified fraction. These studies lead to the identification of a number of RNA-binding and mitochondria-associated proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), Y-box protein 1 (YB-1) and Parkinson disease protein 7 (PARK7/DJ-1) respectively. FUS/TLS and PARK7/DJ-1 are genes that have been previously linked to inherited cases of neurodegenerative disorders such as Amyotrophic lateral sclerosis and Parkinson’s disease, respectively, while YB-1 has been shown to localize with and regulate the translation of nuclear-encoded mitochondrial mRNAs. 

Validation using western blot analyses confirmed the presence of the candidate proteins in the COXIV-zipcode affinity purified complexes from SCG axonal lysates. Immunohistochemical studies show that FUS/TLS, YB-1 and PARK7/DJ-1 co-localize with mitochondrial and cytoskeletal proteins in SCG axons. RNA immunoprecipitation studies with FUS/TLS, YBX-1 and PARK7/DJ-1 antibodies performed on both axonal and cell-body lysates show enrichment of endogenous COXIV transcripts in FUS/TLS, YB-1 and PARK7/DJ-1 immunopurified fractions. siRNA-mediated downregulation of FUS/TLS and YB-1 levels in the cell-body showed a significant decrease in the levels of axonal COXIV mRNA. Interestingly, knockdown of YB-1 also led to a significant reduction in COXIV mRNA transcripts in the cell-bodies, while no change in cell-body COXIV mRNA levels were observed after FUS downregulation. These results suggest differential regulation of COXIV mRNA by FUS/TLS and YB-1. 

Taken together, using this MS-coupled RNA affinity pulldown approach we have started to define the ribonucleoprotein complexes that regulate the trafficking and local expression of the COXIV mRNA in the axon. Our studies suggest that COXIV mRNA trafficking and local translation may be regulated via multiple protein-protein interactions between various RNA-binding proteins.

Biography: 

Dr. Kaplan received his B.A. and M.S. degrees from Hofstra University and his Ph.D. degree in cellular and developmental biology from Cornell University. After completing postdoctoral training in molecular neurobiology at the Andrus Gerontology Center, University of Southern California, he joined the faculty of the Department of Anatomy and Cell Biology of the Cornell University Medical College. In 1983, he moved to the Western Psychiatric Institute and Clinic, University of Pittsburgh Medical Center, where he became Director of the Molecular Neurobiology and Genetics Program. In 1996, he relocated to the NIMH as Associate Director of Fellowship Training in the Division of Intramural Research. Dr. Kaplan’s laboratory is studying the subcellular compartmentation of neuronal gene expression.

 



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