Poster abstracts

Poster number 36 submitted by Geeta Palsule

Biogenesis and functional consequences of the intronic Drosophila RNase P RNA

Geeta Palsule (The Ohio state University, Molecular Cellular Developmental Biology Program), Lien B. Lai (The Ohio state University, Department of Chemistry and Biochemistry ), Venkat Gopalan (The Ohio state University, Department of Chemistry and Biochemistry ), Amanda Simcox (The Ohio state University, Department of Molecular Genetics)

Abstract:
RNase P, an essential and conserved ribonucleoprotein that catalyzes removal of the 5' leader from pre-tRNAs, is comprised of a catalytic RNA (RNase P RNA, RPR) and a variable number of proteins (1). In eukaryotes, RPR was considered a canonical RNA Pol III-regulated transcript, until we discovered that in insects and crustaceans it is embedded in an intron of a Pol II recipient gene and lacks signals for Pol III transcription. In Drosophila, we showed that RPR is transcribed as part of a Pol II-regulated recipient gene transcript (2). To enable this switch in transcriptional control, which occurred ~500 mya, we hypothesize that nucleases involved in processing other ncRNAs were coopted to trim the 5' and 3' ends of the RPR’s recipient intron and generate the mature RPR. To identify these nucleases, I used RNAi knockdown of candidates in Drosophila cells and examined the impact on RPR biogenesis. My results suggest that XRN2 and the exosome are involved in maturation of RPR. Interestingly, XRN2, a 5' to 3' exonuclease, affects both 5' and 3' processing. I am currently investigating how 5' and 3' end processing are coupled by determining if XRN2 recruits factors to the 3' end and/or resolves a secondary structure in the RNA to reverse a block on 3' processing. To determine whether the mode of transcription has functional consequence(s), I am engineering transgenes regulated by an ancestral-type, Pol III promoter. Purification of RNase P from cells expressing a transgene shows that Pol III-derived RPR co-purifies with the holoenzyme. Preliminary results from pre-tRNA cleavage assays suggest that the Pol III-derived RPR may be active. I am using CRISPR/Cas9 genome editing to generate a fly solely expressing a Pol III-regulated RPR, as this will enable a phenotypic analysis at the molecular, cellular and organismal levels. Collectively, these results will determine whether there is a link between transcriptional control, biogenesis, and function in Drosophila RPR.

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 (2010).
2. Manivannan, S. N., Lai, L. B., Gopalan, V. & Simcox, A. Transcriptional control of an essential ribozyme in Drosophila reveals an ancient evolutionary divide in animals. PLoS Genet. 11, e1004893 (2015).

Keywords: RNase P, RNA processing, Transcriptional regulation