Oligomerization properties of fragile-X mental-retardation protein (FMRP) and the fragile-X-related proteins FXR1P and FXR2P

The absence of fragile-X mental-retardation protein (FMRP) results in fragi le-X syndrome. Two other fragile-X-related (FXR) proteins have been describ ed, FXR1P and FXR2P, which are both very similar in amino acid sequence to FMRP. Interaction between the three proteins as well as with themselves has been demonstrated. The FXR proteins are believed to play a role in RNA met abolism. To characterize a possible functional role of the interacting prot eins the complex formation of the FXR proteins was studied in mammalian cel ls. Double immunofluorescence analysis in COS cells over-expressing either FMRP ISO12/FXR1P or FMRP ISO12/FXR2P confirmed heterotypic interactions. Ho wever, Western-blotting studies on cellular homogenates containing physiolo gical amounts of the three proteins gave different indications. Gel-filtrat ion experiments under physiological as well as EDTA conditions showed that the FXR proteins were in complexes of > 600 kDa, as parts of messenger ribo nuclear protein (mRNP) particles associated with polyribosomes. Salt treatm ent shifted FMRP, FXR1P and FXR2P into distinct intermediate complexes, wit h molecular masses between 200 and 300 kDa. Immunoprecipitations of FMRP as well as FXR1P from the dissociated complexes revealed that the vast majori ty of the FXR proteins do not form heteromeric complexes. Further analysis by [S-35]methionine labelling in vivo followed by immunoprecipitation indic ated that no proteins other than the FXR proteins were present in these com plexes. These results suggest that the FXR proteins form homo-multimers pre ferentially under physiological conditions in mammalian cells, and might pa rticipate in mRNP particles with separate functions.