Molecular Characterization of the Human RAI1 Promoter

Smith-Magenis syndrome (SMS) is a type of mental retardation disorder associated with a deletion of DNA from chromosome 17p11.2. It affects approximately18,000 individuals in the US alone. Major phenotypes are associated with a mutation in the Retinoic Acid-Inducible gene-1 (RAI1) and include: developmental delay, repetitive behaviors, short stature, and hoarse voice. Studying the molecular mechanisms that lead to SMS will help promote earlier detection of the disorder and will help develop a better treatment plan for patients. The purpose of this project was to characterize the promoter of RAI1. Studying the promoter of RAI1 will help determine the normal expression of the gene providing clues to its normal function and providing insight into why the lack of RAI1 leads to SMS. The method used for characterizing promoter activity was the reporter gene assay. The goal of this assay was to determine if previously cloned putative promoter fragments contained any promoter activity. The putative RAI1 promoter was cloned in front of the firefly luciferase reporter gene in the pGL3 plasmid and transiently transfected into the mammalian cell line HEK293. The results we obtained suggest that the putative promoter fragments did not contain any promoter activity. In order to identify new putative promoter fragments, 5’RLM-RACE was used on RNA isolated from HEK293 cells. The purpose of the procedure was to identify the 5’ end of the RAI1 transcript. If successfully cloned, the sequence of this fragment could be used to align with the human genome sequence and locate potential promoter elements. These studies are currently underway