The objectives of this proposal are to understand the basic biochemical and molecular principles which govern transcription and post-transcriptional RNA processing in trypanosomes. Several interesting peculiarities of trypanosomes are to be studied in this work: first, identification of the true start of transcription of protein-coding genes, the polymerases responsible for VSG transcription, and the dissection of trans-splicing which occurs obligatorily on mRNAs. Following in the course of extensive published work from this laboratory, 1) in vivo protein-encoding nascent RNA polymerase II transcripts will be isolated and characterized with respect to their start sites and the presence of cap structures; 2) antibodies to each of the four RNA polymerase major subunit genes will be prepared and used to isolate transcription complexes after cross-linking of nascent RNA to proteins by UV irradiation, allowing the identification of the polymerases responsible for transcription; 3) in vitro reconstitution of trans-splicing will be attempted using either cell extracts or biochemically purified components implicated in trans-splicing. SL and U-snRNPs comprising these particles will be characterized and reagents developed for identifying their component RNAs and proteins. This will be employed in the analysis of trans-splicing complexes formed in vivo and in vitro.
Showing the most recent 10 out of 31 publications
