Isolation of an FMRP-associated messenger ribonucleoprotein particle and identification of nucleolin and the fragile X-related proteins as componentsof 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, FMR P, is a member of a gene family that also contains the fragile X-related pr oteins, FXR1P and FXR2P. FMRP has been shown to be a nucleocytoptasmic shut tling protein that selectively binds a subset of mRNAs, forms messenger rib onucleoprotein (mRNP) complexes, and associates with translating ribosomes. Here we describe a cell culture system from which we can isolate epitope-t agged FMRP along with mRNA, including its own message, and at least six oth er 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 s pectrometry. The presence of nucleo;in is confirmed by both reactivity with a specific antiserum as well as reverse coimmunoprecipitation where antinu cleolin antiserum immunoprecipitates endogenous FMRP from both cultured cel ls and mouse brain. The identification of nucleolin, a known component of o ther mRNPs, adds a new dimension to the analysis of FMRP function, and the approach described should also allow the identification of the remaining un known proteins of this FMRP-associated mRNP as well as the other bound mRNA s.