The importance of FMRP (Fragile X Mental Retardation Protein) functional domains.
Fragile X mental retardation protein, FMRP, is absent in Fragile X patient. It has been shown that FMRP mutations lead to Fragile X Syndrome, which is the second most common genetically-inherited mental impairment. Fragile X Syndrome is observed in one in four thousand males, and one in every eight thousand females. The name Fragile X clearly describes the unstable X chromosome in both males and females. The X chromosome in a diagnosed Fragile X person contains an abnormal quantity of the trinucleotide sequence, CGG; which causes the genetic silencing of the FMR1 gene. The mutation of this, located on chromosome (X) q27.3, triggers the outcome of the Fragile X Syndrome FMRP. It is believed to be a repressor which may have a relation with mRNAs at the base of dendrites spines in neurons. Our research deals with RNA methylation in the RGG box. The first insight into the functions of FMRP came from sequence analyses that suggested the presence in FMRP of three potential RNA-binding motifs: an Arg-Gly-Gly sequence (RGG box) and two K-homology (KH) modules. RGG boxes are believed to promote nonspecific binding to nucleic acids through electrostatic interactions, whereas KH modules occur in single or multiple copies in more than 100 RNA-binding proteins and seem to recognize RNA in a sequence-specific way. Our main question is: does methylation affects RNA binding in FMRP? We changed an arginine 538 and 543 to lysine in order to cause a methylation. This gave us the opportunity of observing if this methylation can cause a positive or negative effect on RNA binding. We have methylated the 538 and 543 near the RGG box. This cell lines, LM (TK -),were used because it exhibits a low FMRP expression. The LM (TK-) demonstrated some FMRP expression in order to answer our main question if methylation has an effect on RNA binding in FMRP. We lysed the cells and screen them doing the Western Blot Technique. FMRP was expressed in the transfected cell lines. This happened after three weeks of harvest. The first week did not reveal any expression upon screening. We had a negative vector control and a wild type to compare FMRP expression. We identified clones expressing arginine to lysine substitution in 538 and 543. Our goal was to identify the role of methylation in FMRP function. We used site-directed mutagenesis to change candidate arginine to lysine. Our summer project was to change arginine 545 to a lysine and to screen cell expression methylated at 538 and 543. We identified clones expressing arginine to lysine substitution at 538 and 543 which will be analyzed for their ability to be methylated.
School:
University of Illinois at Urbana-Champaign
Research Advisor:
Dr. Stephanie Ceman
Department of Research Advisor:
Cell and Structural Molecular Biology
Year of Publication:
2004