tRNA m¹G9 modification depends on substrate-specific RNA conformational changes induced by the methyltransferase Trm10

Abstract

The methyltransferase Trm10 modifies a subset of tRNAs on the base N1 position of the 9th nucleotide in the tRNA core. Trm10 is conserved throughout Eukarya and Archaea, and mutations in the human gene (TRMT10A) have been linked to neurological disorders such as microcephaly and intellectual disability, as well as defects in glucose metabolism. Of the 26 tRNAs in yeast with guanosine at position 9, only 14 are substrates for Trm10. However, no common sequence or other posttranscriptional modifications have been identified among these substrates, suggesting the presence of some other tRNA feature(s) which allow Trm10 to distinguish substrate from nonsubstrate tRNAs. Here, we show that substrate recognition by Saccharomyces cerevisiae Trm10 is dependent on both intrinsic tRNA flexibility and the ability of the enzyme to induce specific tRNA conformational changes upon binding. Using the sensitive RNA structure-probing method SHAPE, conformational changes upon binding to Trm10 in tRNA substrates, but not nonsubstrate, were identified and mapped onto a model of Trm10-bound tRNA. These changes may play an important role in substrate recognition by allowing Trm10 to gain access to the target nucleotide. 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.

Diversity in Biological Function and Mechanism of the tRNA Methyltransferase Trm10

“…Recently, we have obtained some insight into at least one of the factors that seems to be associated with tRNA selection by Trm10 in vitro . The selective 2′-OH acylation analyzed by primer extension (SHAPE) reactivity of five in vitro transcribed tRNAs was evaluated, comparing the observed reactivities of the free tRNA to the tRNA bound by Trm10 . The studied transcripts included two in vivo substrates of S. cerevisiae Trm10, tRNA Gly GCC and tRNA Trp CCA , a tRNA only modified in vitro , tRNA Val UAC , and two tRNAs that are not modified by Trm10 in vitro or in vivo , tRNA Leu CAA and tRNA Ser UGA .…”

“…The selective 2′-OH acylation analyzed by primer extension (SHAPE) reactivity of five in vitro transcribed tRNAs was evaluated, comparing the observed reactivities of the free tRNA to the tRNA bound by Trm10. 41 The studied transcripts included two in vivo substrates of S. cerevisiae Trm10, tRNA Gly GCC and tRNA Trp CCA , a tRNA only modified in vitro, tRNA Val UAC , and two tRNAs that are not modified by Trm10 in vitro or in vivo, tRNA Leu CAA and tRNA Ser UGA . 1 SHAPE data revealed that when Trm10 binds to a catalytically competent substrate tRNA, the D-stem becomes more flexible, potentially to allow the enzyme access to the core G9 position for methylation.…”

Investigations of Single-Subunit tRNA Methyltransferases from Yeast

“…Recently, intrinsic tRNA flexibility and enzyme-induced specific tRNA conformational change were found to be key factors for the discrimination of substrate between structurally similar tRNA species by Trm10p for methylation [124]. The conformational changes of substrate tRNA observed by using a sensitive RNA structure-probing method mainly lead to the increased reactivity in the D loop and decreased reactivity in the anticodon loop, which presumably position the target nucleotide in the binding pocket of Trm10p.…”