Poster abstracts

Poster number 94 submitted by Abigail Kelly

Investigating tRNA maturation by Protein-Only RNase P (PRORP) & the Elucidating the Role of Interacting Methyltransferases in Arabidopsis thaliana

Abigail Kelly (University of Michigan), Kip Kaitany (University of Michigan), Markos Koutmos (University of Michigan)

Abstract:
Ribonuclease Proteins (RNase Ps) are a class of enzymes responsible for cleaving the 5' leader sequence of premature tRNAs (pre-tRNA). In 2008, a subclass of RNase P was definitively discovered in the human mitochondria, termed protein-only RNase Ps (PRORPs), which were not catalytically driven by RNA. The complex consisted of PRORP, the catalytic center, and two accessory proteins required for efficient cleavage- TRMT10C, a methyltransferase, and SDR5C1, a dehydrogenase. Later, single subunit PRORPs were discovered in non-metazoan eukaryotes. Much of what we know about these enzymes stems from studies in model plant organisms such as Arabidopsis thaliana (At). While these single subunit PRORPs do not contain accessory proteins like Metazoan mitochondrial (mt)-RNase Ps, it was recently discovered that At-PRORP2, a nuclear enzyme, interacts with methyltransferases At-TRM1A and At-TRM1B. Knockout of both methyltransferases are lethal to plant growth and pre-tRNA processing. I am conducting gel-based cleavage assays and fluorescence polarization to determine kinetic data for different At-tRNAs, as well as using fluorescence polarization to determine thermodynamic behavior. I aim to discover if the enzyme has a sequence or structural preference for cleavage and binding, as some of the At-substrates have very different kobs and KD, values. Additionally, I purified At-TRM1A and am adding it to my FP cleavage assays to see what effect it has on cleavage. It could potentially act as a chaperone, helping the At-tRNAs fold properly as cleavage is occurring. My project aims to better elucidate the PRORP interactome and serves as a model for other PRORP systems.

References:
Arrivé, M.et al. Nature Plants 2023, 9 (12), 2031–2041.
Rossmanith & Robert Karwan. Biochemical and Biophysical Research Communications, 1998, 247(2), 234-241.
Bhatta et al. Nature Structural & Molecular Biology, 2021 28, 713–723.

Keywords: RNase P, tRNA modifying enzyme, kinetics