Defining the mechanism of substrate recognition by the tRNA methyltransferase Trm10 Restricted; Files & ToC

Abstract

RNA modifications are central to proper RNA function and are highly conserved across all kingdoms of life. Of all major RNA classes, transfer RNAs (tRNAs) are the most highly modified with each tRNA molecule containing an average of 13 out of the 94 known modifications. Trm10 (TRMT10A in humans) is a tRNA methyltransferase that is part of the SpoU-TrmD (SPOUT) family of enzymes and is evolutionarily conserved. Trm10 modifies a subset of tRNAs on the base N1 position of guanosine at the ninth nucleotide in the core region. Mutations in the TRMT10A gene have been linked to neurological disorders, such as microcephaly and intellectual disability, as well as defects in glucose metabolism. However, despite the clear biomedical importance of TRMT10A and the tRNA methylation it incorporates, there is still a large gap in our understanding of how this enzyme accurately recognizes its specific substrates to generate the pool of correctly modified tRNAs that is essential for normal cell function. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10 and no common sequence or other posttranscriptional modifications have been identified among these substrates. These observations suggest the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Additionally, little is known about the specific interactions between Trm10 and tRNA that allow for this unique substrate specificity. Here, I show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on the ability of the enzyme to induce specific conformational changes to the tRNA upon binding which allow Trm10 to gain access to the target nucleotide. I also use cryogenic electron microscopy (cryo-EM) to generate a 3D reconstruction of the Trm10-tRNA complex which is the first structural snapshot of a monomeric SPOUT methyltransferase bound to its substrate in the absence of any additional binding partners. Our results highlight a novel mechanism of substrate recognition by a conserved tRNA-modifying enzyme. Further, these studies reveal a strategy for substrate recognition that may be broadly employed by tRNA-modifying enzymes which must distinguish between structurally similar tRNA species. 

Table of Contents

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Palabra Clave	SPOUT methyltransferase Trm10 tRNA methyltransferase cryo-em SHAPE RNA modifications
Committee Chair / Thesis Advisor	Anita H. Corbett, Emory University Eric A. Ortlund, Emory University Christine M. Dunham, Emory University Homa Ghalei, Emory University
Committee Members	Graeme L. Conn, Emory University

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