Activity reconstitution of Kre33 and Tan1 reveals a molecular ruler mechanism in eukaryotic tRNA acetylation

Author:

Ma Chun-Rui1,Liu Na12,Li Hong3,Xu Hong2,Zhou Xiao-Long14ORCID

Affiliation:

1. Key Laboratory of RNA Innovation, Science and Engineering, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences , 320 Yue Yang Road , Shanghai  200031 , China

2. International Peace Maternity & Child Health Hospital, School of Medicine, Shanghai Jiao Tong University , 910 Heng Shan Road , Shanghai  200030 , China

3. Core Facility of Molecular Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences , 320 Yue Yang Road, Shanghai  200031 , China

4. Key Laboratory of Systems Health Science of Zhejiang Province, School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences , Hangzhou  310024 , China

Abstract

Abstract RNA acetylation is a universal post-transcriptional modification that occurs in various RNAs. Transfer RNA (tRNA) acetylation is found at position 34 (ac4C34) in bacterial tRNAMet and position 12 (ac4C12) in eukaryotic tRNASer and tRNALeu. The biochemical mechanism, structural basis and functional significance of ac4C34 are well understood; however, despite being discovered in the 1960s and identification of Kre33/NAT10 and Tan1/THUMPD1 as modifying apparatuses, ac4C12 modification activity has never been reconstituted for nearly six decades. Here, we successfully reconstituted the ac4C12 modification activity of yeast Kre33 and Tan1. Biogenesis of ac4C12 is primarily dependent on a minimal set of elements, including a canonical acceptor stem, the presence of the 11CCG13 motif and correct D-arm orientation, indicating a molecular ruler mechanism. A single A13G mutation conferred ac4C12 modification to multiple non-substrate tRNAs. Moreover, we were able to introduce ac4C modifications into small RNAs. ac4C12 modification contributed little to tRNA melting temperature and aminoacylation in vitro and in vivo. Collectively, our results realize in vitro activity reconstitution, delineate tRNA substrate selection mechanism for ac4C12 biogenesis and develop a valuable system for preparing acetylated tRNAs as well as non-tRNA RNA species, which will advance the functional interpretation of the acetylation in RNA structures and functions.

Funder

National Key Research and Development Program of China

Natural Science Foundation of China

Strategic Priority Research Program of the Chinese Academy of Sciences

Committee of Science and Technology in Shanghai

CAS Project for Young Scientists in Basic Research

Publisher

Oxford University Press (OUP)

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