期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2022
卷号：119
期号：10
DOI：10.1073/pnas.2119529119
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Understanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA. Here, we focus on a cytoplasmic tRNA
Arg
UCU that is expressed specifically in neurons and, when harboring a particular point mutation, contributes to neurodegeneration in mice. Our results suggest that this mutation favors stable alternative structures that are not cleaved by mouse ribonuclease P and motivate a paradigm that may help to understand the molecular basis for disease-associated mutations in other tRNAs.
n-Tr20 is a neuron-specific, cytoplasmic transfer RNA
Arg
UCU (tRNA
Arg
UCU) isodecoder that affects seizure susceptibility, neuronal excitability, and translation signaling in mice. In addition, the C50U substitution in
n-Tr20 (
n-Tr20
C50U
), which is found in the widely used C57BL/6J (B6J) inbred mouse line, contributes to neurodegeneration by epistatic interactions with mutations in
Gtpbp1 or
Gtpbp2, two factors that rescue ribosomal stalling. The brains of B6J mice have high levels of immature and low levels of mature
n-Tr20
C50U
, implicating defective tRNA biogenesis as the basis for neuronal dysfunction, a hypothesis tested in this study. We demonstrate that partially purified mouse brain ribonuclease P, the endonuclease responsible for 5′ maturation of tRNAs, exhibits at 1 mM magnesium a 20-fold lower apparent cleavage rate for processing the
n-Tr20
C50U
precursor than the wild-type precursor. Thermal denaturation studies unexpectedly revealed that substituting the native C
50-G
64 base pair with the U
50-G
64 wobble pair in
n-Tr20
C50U
increased the T
m by as much as 8 °C, with the magnitude of the change dependent on [Mg
2+]. Moreover, results from thermal denaturation, native gel electrophoresis,
1H nuclear magnetic resonance spectroscopy, and kinetic studies collectively support the presence of stable alternative folds for
n-Tr20
C50U
that may also be sampled transiently and in low abundance by the wild-type tRNA. These findings suggest that mutation-driven restructuring could foster nonnative folds and that conformational toggling of tRNAs poses an intrinsic risk for dysfunction when point mutations selectively stabilize nonnative states. This model may be applicable for understanding the molecular basis of other diseases that are associated with tRNA mutations.
