Poster abstracts
Poster number 64 submitted by Markos Koutmos
tRNA processing in human mitochondria
Agnes Karasik (Department of Biological Chemistry, USU), Markos Koutmos (Departments of Chemistry and Biophysics, University of Michigan)
Abstract:
Mitochondrial diseases affect 1 in every 4000 children born in the US. Despite their frequency, the underlying mechanisms of most mitochondrial diseases are unknown. The majority (>50 %) of mitochondrial diseases are linked to mutations in mitochondrial tRNA (mt-tRNA) genes, which make up only ~10% of the human mitochondrial genome. tRNAs are extensively processed and these maturation steps can be critical for physiological function. The first step in mt-tRNA processing is thought to be the 5’ end maturation of tRNAs by the three-component mitochondrial RNase P protein complex (MRPP-complex). The MRPP complex is comprised of MRPP1 (methyltransferase), MRPP2 (dehydrogenase), and MRPP3 (catalytic nuclease subunit, also referred to as Protein Only RNase P, PRORP). While there is an emerging picture of the importance of the human MRPP-complex to human health, little is known about how the MRPP-complex functions to process precursor mt-tRNAs (pre-mt-tRNAs). Our work demonstrates how patient mutations in pre-mt-tRNAs alter their structure and impact the ability of mitochondrial tRNA maturation enzymes to properly mature mt-tRNAs.
We investigated the binding and processing of 11 human pre-mt-tRNAs with disease-causing mutations by the MRPP-complex using a combination of binding and single turnover assays. We find that a subset of mutations reduces substrate binding to the MRPP-complex. Interestingly, all of the mutations that we investigated are inefficiently processed by the MRPP-complex. We posit that this may be due to alterations in pre-mt-tRNA folding and structure because most of the mutant tRNAs showed altered UV melting profiles. Additionally, our data indicate that some pre-tRNAs require further processing/modification(s) and/or an additional as of yet unidentified protein partner to be processed by the MRPP-complex. We find that pre-mt-tRNAVal is bound ~100 times tighter and processed ~10 times more efficiently by the MRPP-complex when the methyl donor and MRPP1 substrate (SAM) is present in our assays, suggesting that methylation of pre-tRNAVal m1A9 is required for efficient 5’ end cleavage. Taken together, our work reveals the first direct evidence for the role of MRPP1 in the MRPP-complex, and also points to the role of MRPP-complex in human disease.
Keywords: tRNA, processing, mitochondria