Poster abstracts

Poster number 43 submitted by Rachel Robertson

The folding and structural dynamics of a tRNA-Arg-UCU isodecoder associated with neurodegeneration

Rachel M. Robertson (Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Ehsan Akbari, Danielle Wampler (Department of Physics, The Ohio State University, Columbus, OH 43210; Center for RNA Biology, Department of Physics, The Ohio State University, Columbus, OH 43210), Kumar Vaibhav (Biophysics Graduate Program, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210), Sebastian Glatt (Malopolska Centre of Biotechnology MCB, Jagiellonian University, Krakow, Poland and Department of Biological Sciences and Pathobiology, University of Veterinary Medicine, Vienna, Vienna, Austria), Ralf Bundschuh (Center for RNA Biology, Department of Chemistry and Biochemistry, Division of Hematology, Department of Physics, The Ohio State University, Columbus, OH 43210), Michael G. Poirier, Venkat Gopalan (Department of Physics, The Ohio State University, Columbus, OH 43210; Center for RNA Biology, Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210)

Abstract:
One of the tRNA-Arg-UCU isodecoders in mice (n-Tr20) and humans (tRNA-Arg-TCT-4-1) is specifically expressed in neurons. In mice, the C50U mutation in n-Tr20 leads to neurodegeneration when associated with the concomitant 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-Tr20-C50U leads to ribosomal stalling, which is accentuated by loss of GTPBP (1). We previously showed that RNase P-mediated 5'-processing of pre–n-Tr20-C50U in vitro is severely hampered and likely accounts for the in vivo accumulation of this precursor (2). We postulated that n-Tr20 samples both native and non-native conformations/ensembles, and that defective 5'-processing of pre-n-Tr20-C50U 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-Tr20-C50U. For our force-spectroscopy studies, we first prepared n-Tr20 and n-Tr20-C50U each with 5'- and 3'-single-stranded extensions to enable their respective annealing to DNA tethers designed to have overhangs complementary to the engineered extensions. We used native polyacrylamide gel electrophoresis to confirm that the stable conformations adopted by n-Tr20 and n-Tr20-C50U are not altered by inclusion of the 5'- and 3'-extensions. The force-extension curves show that n-Tr20 and n-Tr20-C50U differ with respect to the type and reversibility of the transitions. Moreover, quantitative predictions of the force-extension behavior of n-Tr20-C50U are consistent with the optical tweezer force-extension curves and the ability of the mutant to sample secondary structures different from the tRNA cloverleaf structure. These predicted alternative tertiary structures are supported by rigid-body fitting into cryo-EM maps of n-Tr20 and n-Tr20-C50U (3). 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
3. Dutt et al. (2026) Nucleic Acids Res. 54: gkaf1411

Keywords: tRNA, Optical tweezers, Conformational dynamics