Poster abstracts

Poster number 86 submitted by Rachel Robertson

Investigating the folding and dynamics of a tRNAArgUCU isodecoder associated with neurodegeneration

Rachel M. Robertson (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University), Ehsan Akbari, Danielle Wampler (Center for RNA Biology, Department of Physics, The Ohio State University), Henry Arthur (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University), Ralf Bundschuh (Center for RNA Biology, Department of Physics, The Ohio State University), Michael G. Poirier (Department of Physics, The Ohio State University), Venkat Gopalan (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University)

Abstract:
One of the tRNAArgUCU isodecoders in mice (n-Tr20) and human (tRNA-Arg-TCT-4-1) is specifically expressed in neurons. In mice, the C50U mutation in n-Tr20 leads to neurodegeneration when associated with a loss of guanosine-5'-triphosphate binding protein 1/2 (GTPBP1/2), a ribosomal release factor. In these double-mutant mice, the accumulation of pre–n-Tr20C50U leads to ribosomal stalling, which is accentuated by loss of GTPBP.1 We showed that RNase P-mediated 5'-processing of n-Tr20C50U in vitro is severely hampered and likely to account for the in vivo accumulation of pre–n-Tr20C50U.2 We postulated that n-Tr20 samples both native and non-native conformations, and that defective 5'-processing of pre-n-Tr20C50U is due to the mutant tRNA favoring a fold that is not recognized by RNase P. Here, we employed single-molecule force spectroscopy and computational modeling to investigate the folding and conformational dynamics of n-Tr20 and n-Tr20C50U. For our force-spectroscopy studies, we first prepared n-Tr20 and n-Tr20C50U each with 5'- and 3'-single-stranded extensions to enable their annealing to DNA tethers designed to have overhangs complementary to the engineered extensions. We used native polyacrylamide gel electrophoresis (PAGE) to confirm that the stable conformations adopted by n-Tr20 and n-Tr20C50U are not altered by inclusion of the 5'- and 3'-extensions. The force-extension curves show that n-Tr20C50U differs from n-Tr20 in having only one instead of two reversible transitions. Moreover, quantitative predictions of the force-extension behavior of n-Tr20C50U are consistent with the mutant sampling secondary structures different from the tRNA cloverleaf structure. These results are consistent with our previous thermal melt studies which showed that n-Tr20C50U is more stable than n-Tr20 and native PAGE experiments which revealed that n-Tr20C50U and n-Tr20 adopt different conformational ensembles in the presence of magnesium.2 Collectively, our findings help understand how a single point mutation impacts the conformational ensemble and dynamics of n-Tr20.

References:
1. Ishimura et al. (2014) Science 345: 455
2. Lai et al. (2022) Proc. Natl. Acad. Sci. USA 119: e2119529119

Keywords: tRNA, Optical tweezers, Conformational dynamics