Defining the mechanism of substrate recognition by the tRNA methyltransferase Trm10 Open Access
Strassler, Sarah (Fall 2023)
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
Chapter 1: Introduction. 1
Gene Expression. 2
The Roles of RNA During Translation. 2
Regulating Translation through RNA Modifications. 3
tRNA. 4
tRNA-Modifying Enzymes. 4
tRNA Modifications. 5
The tRNA Methyltransferase Trm10. 6
Biological Relevance of Trm10. 6
Trm10 Enzymes in Humans. 7
The SPOUT Family of Methyltransferases. 7
Trm10 Structure. 8
Research Goals. 10
Figures. 12
Figure 1.1 The Central Dogma. 12
Figure 1.2 tRNA Secondary and Tertiary Structure. 13
Figure 1.4 The Structure of Trm10. 14
Figure 1.5 Overview of Research Objectives. 15
References. 16
Chapter 2: Tied up in knots: Untangling substrate recognition by the SPOUT methyltransferases. 21
Abstract 22
Introduction. 22
Phylogenetic Analysis of the SPOUT Methyltransferase Superfamily. 26
SPOUT Methyltransferase Structure, SAM Binding, and Domain Organization. 29
Substrate Recognition and Modification by SPOUT Methyltransferases. 33
Ribose 2’-O-methylating SPOUT RNA methyltransferases. 34
Base-modifying SPOUT RNA methyltransferases. 40
Sfm1: A protein-modifying SPOUT methyltransferase. 48
Role of Molecular Conformational Dynamics in Substrate Recognition and Modification. 49
Bent SAM conformation. 50
Protein dynamics. 52
Protein-induced RNA conformational changes. 53
Conclusions. 57
Acknowledgements. 58
Author Contributions. 59
Figures. 59
Figure 2.1 RNA SPOUT methyltransferase target sites in tRNA and rRNA. 60
Figure 2.2 Phylogenetic analysis of the SPOUT superfamily. 61
Figure 2.3 Overview of SPOUT methyltransferase structure. 63
Figure 2.4 SAM conformations and SAM-binding pockets of representative SPOUT methyltransferases. 64
Figure 2.5 Substrate Recognition and Base-flipping in SPOUT methyltransferase-RNA substrate complexes. 65
Tables. 67
Table 2.1. 67
References. 68
Chapter 3: tRNA m1G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10. 78
Abstract 79
Introduction. 79
Materials and Methods. 82
RNA in vitro transcription and purification. 82
Trm10 expression and purification. 83
NM6 preparation. 83
tRNA SHAPE analysis. 84
Isothermal titration calorimetry. 85
Mass Spectrometry (MS) 86
Fluorescence anisotropy. 86
Electromobility Shift Assay. 87
Trm10 methyltransferase activity assay. 87
Results. 88
SHAPE analysis reveals differences in inherent flexibility of Trm10 substrate and nonsubstrate tRNAs. 88
Trm10 induces specific conformational changes in substrate tRNAs that are not observed in nonsubstrate tRNA. 90
Comparison of tRNA bound to Trm10 and Trm10-KRR highlights conformational changes specifically necessary for methylation. 93
Mapping of SHAPE reactivity onto a Trm10-tRNA model highlights interactions critical for required conformational changes. 96
Discussion. 97
Acknowledgements. 101
Author Contributions. 102
Figures. 103
Figure 3.1 Comparison of modification reaction kinetics for authentic tRNA transcripts and tRNAs embedded within 5’- and 3’-end hairpins. 103
Figure 3.2 SHAPE analysis reveals differences in inherent flexibility of substrate and nonsubstrate tRNAs. 104
Figure 3.3 Trm10 induces specific conformational changes in substrate tRNAs that are not observed in nonsubstrate tRNAs. 105
Figure 3.4 S. cerevisiae Trm10-KRR has similar substrate and cosubstrate binding affinities as the wild-type enzyme but lacks catalytic activity. 106
Fig. 3.5. Comparison of SHAPE reactivities when bound to wild-type Trm10 and Trm10-KRR variant reveals the tRNA conformational changes necessary for methylation. 108
Supplemental Figures. 110
Supplemental Figure S3.1 Binding affinity of tRNAs with Trm10. 110
Supplemental Figure S3.2. Stabilization of the Trm10-tRNA complex using the SAM analog NM6. 111
Supplemental Figure S3.3 Normalized reactivities of tRNAs bound to wild-type Trm10 and Trm10-KRR. 112
Supplemental Figure S3.4 Nucleotide SHAPE reactivities in free and Trm10-bound tRNAs. 113
Supplemental Figure S3.5 SHAPE reactivities of substrate tRNATrp and nonsubstrate tRNALeu bound to wild-type Trm10 in the presence of SAH. 114
Supplemental Figure S3.6 Electromobility shift assay (EMSA) of substrate tRNAGly with wild-type Trm10 and Trm10-KRR. 115
Supplemental Figure S3.7 Comparison of unbound tRNA and KRR-bound tRNA. 116
Supplemental Figure S3.8 Electrostatic surface potential of wild-type Trm10 and Trm10-KRR shown on a Trm10-tRNA model. 117
Supplemental Table. 118
Supplemental Table 3.1 Association constants (KA) for Trm10 binding SAM and SAH determined by ITC. 118
Chapter 4: Cryo-EM structure of the tRNA methyltransferase Trm10 bound to substrate tRNA.. 125
Abstract 126
Introduction. 127
Materials and Methods. 130
Trm10 expression and purification. 130
RNA in vitro transcription and purification. 130
NM6 preparation. 131
Trm10-tRNA complex formation and grid preparation. 131
Screening grid types and preparation conditions. 132
Cryo-EM image collection, processing and analysis. 133
Electromobility shift assay (EMSA) 134
BS3 crosslinking assay. 134
Results. 135
Tilted UltrAuFoil grids provide optimal ice thickness and range of orientations for structural analysis using Cryo-EM.. 135
Trm10-tRNA model shows protein binding to tRNA in a 1:1 ratio. 136
Trm10 makes specific contacts with different regions of the tRNA. 136
Trm10 binds to substrate tRNA in a manner similar to TRMT10C. 137
Trm10 dimerization observed upon binding to substrate tRNA. 138
Discussion. 139
Acknowledgements. 142
Figures. 144
Figure 4.1 Stabilization of the Trm10-tRNA complex using the SAM analog NM6. 144
Figure 4.2 Cryo-EM data collection and 2D classes. 145
Figure 4.3. Trm10-tRNA 3D reconstruction. 146
Figure 4.4 Structure of Trm10 and tRNA modeled into 3D reconstruction. 147
Figure 4.5 Position of S. cerevisiae Trm10 and TRMT10C in relation to tRNA. 148
Figure 4.6 Trm10 dimerization upon binding to substrate tRNA. 149
References. 150
Chapter 5: Discussion. 153
Validate the role of the NTD of Trm10. 156
Define the relevance of a Trm10 dimer interaction during substrate recognition. 157
Characterize the molecular basis for differences in substrate selectivity of S. cerevisiae Trm10 and other family members. 159
Final Remarks. 161
Figures. 163
Figure 5.1 Overview of main research findings. 163
Figure 5.2 Trm10 dimer has a possible role in initiating catalysis. 164
References. 165
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