Identification of FMRP-associated mRNAs using yeast three-hybrid system

Am J Med Genet B Neuropsychiatr Genet. 2008 Sep 5;147B(6):769-77. doi: 10.1002/ajmg.b.30678.

Abstract

Fragile X syndrome, one of the most common forms of inherited mental retardation, results from the absence of the fragile X mental retardation protein (FMRP), which is encoded by the fragile X mental retardation gene 1 (FMR1). FMRP is an RNA-binding protein involved in translational regulation of targeted mRNAs. Identification of targeted mRNAs associated with FMRP is important to understand the function of FMRP and the pathogenic basis of the fragile X syndrome. Employing a yeast three-hybrid system and a human fetal hippocampus cDNA library, we identified 22 candidate target mRNAs, and 18 of them were confirmed to be associated with FMRP in vitro by gel retardation. Some of these mRNAs code for structural proteins, enzymes or proteins involved in cellular processes, especially in the development and function of neural system. To further investigate the role of FMRP in regulating targeted gene expression, we analyzed the expression profile of TXNRD1, one of the candidate mRNAs, after knocking down the expression of endogenous FMRP by siRNA. The results showed that endogenous TXNRD1 translation increased along with depletion of FMRP, which suggested FMRP negatively regulates TXNRD1 translation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Fragile X Mental Retardation Protein / genetics
  • Fragile X Mental Retardation Protein / metabolism*
  • Gene Expression Regulation
  • Gene Library
  • HeLa Cells
  • Hippocampus / chemistry
  • Hippocampus / metabolism
  • Humans
  • Male
  • Mice
  • Mice, Knockout
  • Protein Biosynthesis / genetics
  • RNA, Messenger / isolation & purification*
  • RNA, Messenger / metabolism
  • Thioredoxin Reductase 1 / genetics
  • Thioredoxin Reductase 1 / metabolism
  • Two-Hybrid System Techniques*

Substances

  • FMR1 protein, human
  • RNA, Messenger
  • Fragile X Mental Retardation Protein
  • TXNRD1 protein, human
  • Thioredoxin Reductase 1