ABSTRACT 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 m 1 A58 as a late modification introduced after more initial modifications, such as Ѱ55 and T54 in yeast elongator tRNA Phe . However, previous reports suggested that m 1 A58 is introduced early along the tRNA modification process, with m 1 A58 being introduced on initial transcripts of initiator tRNA i Met , and hence preventing its degradation by the nuclear surveillance and RTD pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m 1 A58 incorporation, we examined the m 1 A58 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 tRNA Phe is directly stimulated by Ѱ55, and that the incorporation of m 1 A58 is directly and individually stimulated by Ѱ55 and T54, thereby reporting on the molecular aspects controlling the Ѱ55 → T54 → m 1 A58 modification circuit in yeast elongator tRNAs. We also show that m 1 A58 is efficiently introduced on unmodified tRNA i Met , and does not depend on prior modifications. Finally, we show that the m 1 A58 single modification has tremendous effects on the structural properties of yeast tRNA i Met , with the tRNA elbow structure being properly assembled only when this modification is present. This rationalizes on structural grounds the degradation of hypomodified tRNA i Met lacking m 1 A58 by the nuclear surveillance and RTD pathways.

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