Poster abstracts

Poster number 38 submitted by Vaishnavi Sidharthan

Characterization and application of Methanocaldococcus jannaschii RNase P, an ancient tRNA processing enzyme.

Vaishnavi Sidharthan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Bela Haifa Khairunisa (Genetics, Bioinformatics, and Computational Biology Graduate Program, Virginia Tech, Blacksburg, VA), Hong Duc Phan (The Ohio State Biochemistry Program, and Center for RNA Biology, The Ohio State University), Biswarup Mukhopadhayay (Department of Biochemistry, Virginia Tech, Blacksburg, VA), Venkat Gopalan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University)

Abstract:
RNase P catalyzes removal of the 5'-leader of precursor-tRNAs (pre-tRNAs). The ribonucleoprotein form of this enzyme is comprised of a catalytic RNase P RNA (RPR) and a variable number of RNase P protein (RPP) cofactors: one in bacteria, up to five in archaea, and up to ten in eukaryotes. Since the five archaeal RPPs have eukaryotic homologs, the simpler composition and biochemical tractability of archaeal RNase P has inspired its use as an experimental surrogate for the larger eukaryotic relative. The recent cryo-EM structure1 of in vitro reconstituted RNase P from Methanocaldococcus jannaschii (Mja, a hyperthermophilic archaeon) has yielded insights into the arrangement of all six subunits and the active site. However, the stoichiometry for one of the RPPs is inconsistent with findings from native mass spectrometry (MS) of in vitro assembled Mja RNase P2. Also, the higher protein content in archaeal/eukaryotic RNase P mirrors the separation of the archaeal/eukaryotic transcriptional machineries from the simpler bacterial version and motivates our investigation of potential crosstalk between RNase P and other cellular machineries3. Towards the use of MS/MS-based proteomics to characterize the subunit make-up, post-synthetic modifications, and the interactome of Mja RNase P, we are leveraging a new method4 for transforming Mja with foreign DNA and constructing in-frame gene deletions or affinity tagging of a target gene. Specifically, we will replace the native Mja RPR with an affinity-tagged variant; we verified that this variant, post-reconstitution with the 5 RPPs, is functionally comparable to the untagged RPR. Also, we are exploring the use of RNase P as a gene knockdown tool5, an important goal given the lack of functional annotation for ~50% of Mja genes and paucity of functional genomics methods (e.g., CRISPR) for use in thermophiles. To this end, we have used in vitro assays to direct our design of external guide sequences, which upon binding to a target mRNA forms a pre-tRNA-like bipartite complex and renders the mRNA a substrate for RNase P cleavage. Results from ongoing pilot studies that target the Mja sulfite reductase mRNA6 will be shared.

References:
1. Wan, F.; Wang, Q.; Tan, J.; Tan, M.; Chen, J.; Shi, S.; Lan, P.; Wu, J.; Lei, M., Nat Commun 2019, 10, 2617.
2. Lai, L. B.; Tanimoto, A.; Lai, S. M.; Chen, W. Y.; Marathe, I. A.; Westhof, E.; Wysocki, V. H.; Gopalan, V., Nucleic Acids Res 2017, 45, 7432-7440.
3. Serruya, R., Orlovetskie, N., Reiner, R., Dehtiar-Zilber, Y., Wesolowski, D., Altman, S., Jarrous, N., Nucleic Acids Res 2015, 43, 5442-5450.
4. Susanti, D.; Frazier, M. C.; Mukhopadhyay, B., Front Microbiol 2019, 10, 1256.
5. Cho, I. M.; Kazakov, S. A.; Gopalan, V., J Mol Biol 2011, 405, 1121-1127.
6. Johnson, E. F.; Mukhopadhyay, B., J Biol Chem 2005, 280, 38776-33786.

Keywords: RNase P, Mja, knockdown tool