Talk abstracts

Talk on Wednesday 04:30-04:45pm submitted by Sathiya Narayanan Manivannan

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

Sathiya N. Manivannan (Department of Molecular Cellular and Developmental Biology), Lien B. Lai (Department of Biochemistry), Venkat Gopalan (Department of Biochemistry), Amanda A. Simcox (Department of Molecular Genetics)

Abstract:
In all three domains of life, RNase P is a ribonucleoprotein (RNP) complex that catalyzes removal of the 5'- leader in precursor tRNAs (pre-tRNAs). The RNase P RNA (RPR) is the catalytic subunit responsible for this Mg²⁺-dependent endonucleolytic cleavage. In silico searches have identified the genes that code for the RPR in several eukaryotes including members of Drosophilidae. Interestingly, the putative RPR gene in Drosophila lies within the second intron of the protein-coding gene CG1746 that has no apparent functional association with RNase P. The RPR gene is in the same orientation as CG1746 and lacks a typical pol III promoter and terminator in its proximal flanking regions. The unusual gene structure of Drosophila RPR motivated us to examine if this RPR is generated by a novel biogenesis mechanism involving splicing of the CG1746 mRNA, and subsequent processing of the intron. Towards uncovering and establishing such a novel RPR biogenesis mechanism, we first sought to prove that the annotated gene indeed codes for RNase P. We tested the authenticity of the intronic RNase P RNA (iRPR) through biochemical characterization of the native RNase P holoenzyme isolated from Drosophila cultured cells. Partially purified RNase P, obtained after tandem anion and cation exchange chromatography of a crude lysate, cleaved a pre-tRNA^Gly substrate accurately compared to Escherichia coli RNase P. Fractions with RNase P activity also contained iRPR as determined by RT-PCR and their activity could be specifically inhibited by an RNA oligonucleotide complimentary to iRPR. Since this evidence supports iRPR as the bona fide Drosophila RPR, we are testing several possible models for its biogenesis. As a first step, we are using a red fluorescent protein (RFP)-based reporter system that carries the second intron of CG1746 to determine if splicing is required for biogenesis of the iRPR and to investigate if all the necessary cis elements are present in the intron.

References:
1. Lai, L.B., Vioque, A., Kirsebom, L.A. & Gopalan, V. Unexpected diversity of RNase P, an ancient tRNA processing enzyme: challenges and prospects. FEBS Lett 584, 287-96 (2009).
2. Piccinelli, P., Rosenblad, M.A. & Samuelsson, T. Identification and analysis of ribonuclease P and MRP RNA in a broad range of eukaryotes. Nucleic Acids Res 33, 4485-95 (2005).

Keywords: Drosophila, RNase P RNA, Splicing