Talk abstracts

Talk on Saturday 10:45-11:00am submitted by Sathiyanarayanan Manivannan

Non-canonical biogenesis of the catalytic RNA subunit of Drosophila RNase P- a tRNA processing enzyme

Sathiya N. Manivannan (MCDB, The Ohio State University), Lien B. Lai (Biochemistry, The Ohio State University), Amanda A. Simcox (Molecular Genetics, The Ohio State University), Venkat Gopalan (Biochemistry, The Ohio State University)

Abstract:
In all three domains of life, the RNA subunit of the ribonucleoprotein form of RNase P is the catalyst responsible for Mg²⁺-dependent endonucleolytic 5′ processing of precursor tRNAs (pre-tRNAs). In silico searches have identified eukaryotic genes that encode the RNase P RNA (RPR). While several eukaryotic RPRs studied thus far (human, yeast and zebrafish) are pol III transcripts, the Drosophila RPR is not detected in a pol III-specific chromatin immuno-precipitation (ChIP) assay. Interestingly, the annotated RPR gene is present in the second intron of the uncharacterized protein-coding gene CG1746. The overall RPR-coding region is highly conserved in all 12 Drosophila species, however, the 5′ and 3′ flanking regions are poorly conserved and lack signature elements of a pol III transcript such as the TATA box and a poly-T terminator. The intronic RPR (iRPR) is expresed throughout development. In biochemical assays, we found that iRPR co-purifies with the active native RNase P holoenzyme from Drosophila cells. Not only did the iRPR alone show activity in vitro, an RNA oligonucleotide complementary to its sequence effectively inhibited the pre-tRNA processing activity of the partially-purified RNase P holoenzyme, confirming that it is the catalytic moiety. To investigate how this iRPR is processed from the spliced intron of CG1746, we have developed a red fluorescent protein (RFP)-based reporter assay. By introducing the intron encoding the iRPR into the RFP-coding region, we have been able to use RFP splicing and expression as a direct monitor of iRPR biogenesis in Drosophila cells. These studies are expected to provide insights into the possible coupling of iRPR biogenesis to pol II transcription and mRNA splicing, and also highlight the regulatory web around this ancient ribozyme.

Keywords: RNase P, Drosophila, Splicing