U2 RNA 3 Processing

Kleinschmidt, Ann

Abstract

The research outlined in this application is designed to determine the parameters important for the 3' processing and cellular localization of U2 RNA, a small nuclear RNA (snRNA) that as a ribonucleoprotein complex (snRNP) participates in the processing of pre-messenger RNAs. It is likely that understanding how the metabolism of U2 RNA can be altered will illuminate features important for RNA metabolism in general. As is true for the control of RNA transcription, control of the subsequent events in RNA metabolism, including processing, nuclear export and RNA turnover can also play a major role in the regulation of the level of particular gene products. It is clear that alterations in RNA processing can lead to health related defects as is the case in certain types of thalassemias. Therefore, it is of interest to understand what types of changes can alter these metabolic events. The proposal is separated into three major projects. THe first investigates how sequence alterations in the U2 RNA precursor molecule affect processing in vivo. This involves generating mutant U2 DNA templates from which 32P-labeled U2 RNA precursors can be transcribed in vitro. This RNA is then introduced into mammalian cells via calcium phosphate-mediated transfection and the extent of processing of the mutant U2 RNAs is determine. The second project is designed to characterize the U2 RNA processing activity. The similarity between the cellular U2 RNA processing activity and a partially purified cytoplasmic activity that accurately processes wild type U2 RNA will be addressed by comparison of the processing characteristics of mutant RNAs in both systems. Further purification of the processing activity will be carried out. The third project analyzes the correlation between U2 RNA 3' processing and its cellular localization. This will be accomplished by introducing various processing deficient U2 RNAs into mammalian cells by electroporation and determining the rate and absolute amount of nuclear transport of these mutants RNAs.