Identification of polyribosomes-associated mRNAs regulated by fragile X mental retardation protein (FMRP) using oligonucleotide microarrays. P. Jin¹, Y. Feng², V. Brown¹, S.T. Warren¹. 1) Howard Hughes Medical Institute and Department of Biochemistry, Pediatrics and Genetics; 2) Department of Pharmacology, Emory University School of Medicine, Atlanta, GA.
   Fragile X syndrome, a common form of inherited mental retardation, is mainly caused by the absence of functional FMRP, the product of FMR1 gene. FMRP is an RNA-binding protein that shuttles between the nucleus and cytoplasm and has been implicated in protein translation as it is found associated with polyribosomes. The functional significance of polyribosomal association is further solidified by the observation that FMRP point mutation, I304N, incorporates into abnormal mRNP particles and does not associate with polyribosomes. However, the downstream target genes, whose translation is directly regulated by FMRP, have not been identified yet. To accomplish this, we use oligonucleotide microarray analysis to identify the mRNAs, whose distribution is changed in the polyribosomal fraction but not at the overall transcriptional level. Using EBV-transformed human lymphoblastoid cell lines, RNA from whole cell lysates and large polyribosomal complexes were pooled to reduce individual variation, from either 5 normal males, 5 fragile X males, or an I304N patient, and subjected to microarray analysis. Compared to normal cells, more than 140 of 35,000 human genes and ESTs show significant change in the polyribosomal fraction from fragile X cells, but only 40 of them have no change at the overall transcriptional level, which suggests that distributions of these genes in polyribosomes may be regulated by FMRP. These results are further supported by finding that 15 of those 40 genes show a similar change in I304N cells. Among them is NAP-22, a cortical cytoskeleton-associated protein, which regulates nerve sprouting and synaptic growth. NAP-22 mRNA can also be co-immunoprecipitated with FMRP from mouse brain, which together suggests that NAP-22 translation is being compromised in the absence of FMRP. This study, for the first time, shows the cellular consequence in the absence of FMRP and should illuminate the physiological functions of FMRP and molecular pathogenesis of fragile X syndrome.