Yared, M.-J. | Search bioRxiv

bioRxiv2026

m1A58 acts as a conformational checkpoint coupling human initiator tRNA maturation to translation initiation

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N1-methyladenosine at position 58 (m1A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m1A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNAiMet) as selectively vulnerable to m1A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNAiMet showed that m1A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m1A58 promotes RNase P-mediated 5'-leader removal and RNase Z-mediated 3'-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m1A58-modified tRNAiMet. Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m1A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.

molecular biology↗

bioRxiv2022

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs

As essential components of the cellular protein synthesis machineries, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of a large number of posttranscriptional chemical modifications. Maturation defaults resulting in lack of modifications in the tRNA core may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. Although modifications are typically introduced in tRNAs independently of each other, several modification circuits have been identified in which one or more modifications stimulate or repress the incorporation of others. We previously identified m1A58 as a late modification introduced after more initial modifications, such as 55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early along the tRNA modification process, with m1A58 being introduced on initial transcripts of initiator tRNAiMet, and hence preventing its degradation by the nuclear surveillance and RTD pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined the m1A58 modification pathways in yeast elongator and initiator tRNAs. For that, we first implemented a generic approach enabling the preparation of tRNAs containing specific modifications. We then used these specifically modified tRNAs to demonstrate that the incorporation of T54 in tRNAPhe is directly stimulated by 55, and that the incorporation of m1A58 is directly and individually stimulated by 55 and T54, thereby reporting on the molecular aspects controlling the 55 [->] T54 [->] m1A58 modification circuit in yeast elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that the m1A58 single modification has tremendous effects on the structural properties of yeast tRNAiMet, with the tRNA elbow structure being properly assembled only when this modification is present. This rationalizes on structural grounds the degradation of hypomodified tRNAiMet lacking m1A58 by the nuclear surveillance and RTD pathways.

biochemistry↗