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PMID: 10567518 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex.

Molecular and cellular biology ·Vol. 19 ·No. 12 ·1999-12-00 ·Pages 7925-32

Ceman S, Brown V, Warren ST

Abstract

The loss of FMR1 expression due to trinucleotide repeat expansion leads to fragile X syndrome, a cause of mental retardation. The encoded protein, FMRP, is a member of a gene family that also contains the fragile X-related proteins, FXR1P and FXR2P. FMRP has been shown to be a nucleocytoplasmic shuttling protein that selectively binds a subset of mRNAs, forms messenger ribonucleoprotein (mRNP) complexes, and associates with translating ribosomes. Here we describe a cell culture system from which we can isolate epitope-tagged FMRP along with mRNA, including its own message, and at least six other proteins. We identify two of these proteins as FXR1P and FXR2P by using specific antisera and identify a third protein as nucleolin by using mass spectrometry. The presence of nucleolin is confirmed by both reactivity with a specific antiserum as well as reverse coimmunoprecipitation where antinucleolin antiserum immunoprecipitates endogenous FMRP from both cultured cells and mouse brain. The identification of nucleolin, a known component of other mRNPs, adds a new dimension to the analysis of FMRP function, and the approach described should also allow the identification of the remaining unknown proteins of this FMRP-associated mRNP as well as the other bound mRNAs.

MeSH Terms
Animals Cell Line Fragile X Mental Retardation Protein Fragile X Syndrome Gene Expression Mice Mice, Knockout Nerve Tissue Proteins/genetics,isolation & purification,metabolism Oligopeptides Peptides/genetics Phosphoproteins/isolation & purification,metabolism Precipitin Tests RNA, Messenger RNA-Binding Proteins/genetics,isolation & purification,metabolism Recombinant Proteins/genetics,isolation & purification,metabolism Ribonucleases/metabolism Ribonucleoproteins/genetics,isolation & purification,metabolism
Chemicals
FXR1 protein, human FXR2 protein, human Fmr1 protein, mouse Nerve Tissue Proteins Oligopeptides Peptides Phosphoproteins RNA, Messenger RNA-Binding Proteins Recombinant Proteins Ribonucleoproteins nucleolin Fragile X Mental Retardation Protein FLAG peptide Ribonucleases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Ceman S
Howard Hughes Medical Institute and Departments of Biochemistry, Pediatrics, and Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Brown V
Warren S T
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.19.12.7925
Published
1999-12-00
Pages
7925-32
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC84877
Subset
IM
Grants
NICHD NIH HHS · P01 HD035576 · United States
NICHD NIH HHS · R37 HD020521 · United States
NICHD NIH HHS · P01HD35576 · United States
NICHD NIH HHS · R37HD20521 · United States
External Links
PubMed source DOI: 10.1128/MCB.19.12.7925 PMC Full Text View journal details
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