Poster abstracts
Poster number 41 submitted by Geeta Palsule
Transcriptional control and biogenesis of intronic RNase P RNA in Drosophila
Geeta Palsule (Molecular Genetics, The Ohio State University ), Lien B. Lai (Chemistry and Biochemistry, The Ohio State University ), Venkat Gopalan (Chemistry and Biochemistry, The Ohio State University ), Amanda Simcox (Molecular Genetics, The Ohio State University )
Abstract:
RNase P, an essential ribonuclease, is present in all domains of life and is required for the removal of 5’ leader sequences from pre-tRNAs. The ribonucleoprotein form of the enzyme is comprised of a catalytic RNA (RNase P RNA, RPR) and a variable number of protein subunits (RNase P Proteins, RPPs). Although RPR is a typical Pol III-regulated gene in most organisms, we found the Drosophila gene is inserted into a recipient gene intron and transcribed by Pol II. By analyzing genomes of other insects and crustaceans, we discovered the genetic change in the regulation of RPR originated in an ancestor of this major group of animals some 500 million years ago. Clearly this dependence on recipient gene transcription and processing from an intron requires a distinct mode of biogenesis for the inserted RPR. I am using a genetic approach to find the factors involved in RPR biogenesis and to test the functional consequence, if any, of the change in transcriptional control. Using reporter genes assayed in Drosophila S2 tissue-culture cells, we found both a splicing-dependent and splicing-independent mode of producing mature RPR. The 5’ end of the pre-RPR is processed by XRN2 and I am currently investigating the exosome and Rexo5 as 3’ end-processing candidates. To investigate the involvement of the nine protein subunits (RPPs) in RPR maturation, I depleted individual RPPs and assayed for mature RPR by northern blot analysis. This indicated several RPPs are required for RPR maturation or stability. Interestingly, however, when I switch RPR transcription back to the ancestral, Pol III-mode, RPR is stable and unaffected when RPPs are depleted. I am using CRISPR/Cas9 to engineer flies with an ancestral, Pol III-regulated gene, so that I can analyze any functional consequences. Overall my work is expected to provide insights into small RNA biogenesis and how the switch in gene transcription contributed to the evolutionary history of RPR.
Keywords: RNase P RNA, Transcriptional control, RNA processing