Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as components of the complex.
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.
Ceman
S
S
Howard Hughes Medical Institute and Departments of Biochemistry, Pediatrics, and Genetics, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Brown
V
V
Warren
S T
ST
D000818:Animals; D002460:Cell Line; D051860:Fragile X Messenger Ribonucleoprotein 1; D005600:Fragile X Syndrome; D015870:Gene Expression; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D012333:RNA, Messenger; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000097796:Nucleolin
D051860:Fragile X Messenger Ribonucleoprotein 1; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D010455:Peptides; D010750:Phosphoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012260:Ribonucleases; D012261:Ribonucleoproteins; D000097796:Nucleolin; C096916:FXR1 protein, human; C096915:FXR2 protein, human; C497684:Fmr1 protein, mouse; C085706:FLAG peptide