1H, 15N chemical shift assignments of the imino groups of yeast tRNAPhe: influence of the post-transcriptional modifications

Abstract

Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In particular, the introduction of post-transcriptional modifications in tRNAs is controlled and influenced by multiple factors. In turn, tRNA biological functions are often modulated by their modifications. Although modifications play essential roles in tRNA biology, methods to directly detect their introduction during tRNA maturation are rare and do not easily provide information on the temporality of modification events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. Here we report the 1 H, 15 N chemical shift assignments of imino groups in three forms of the yeast tRNA Phe differing in their modification content. These assignments are a prerequisite for the time-resolved NMR monitoring of yeast tRNA Phe maturation in yeast extracts.To cite this article: M. Catala et al. Biomolecular NMR Assignments (2020), in press. This is a postprint of an article published in Biomolecular NMR Assignments.

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

“…In addition, a pronounced signal heterogeneity is present in the unmodified tRNA i Met , with weak and strong signals coexisting, while the m 5 C48,49-tRNA i Met spectrum shows a more homogeneous signal profile, with overall stronger signals than in the unmodified tRNA i Met spectrum (Figure 4a,b). Since NMR signals of RNA imino groups are only observed on condition that the imino protons are protected from exchange with the solvent by hydrogen bonding in Watson-Crick or other type of base pairing, the signal heterogeneity present in the unmodified tRNA i Met overall reflects less stable base pairs, as well as a dynamic and less homogeneous folding of this tRNA, compared with the m 5 C48,49-tRNA i Met , or the unmodified yeast tRNA Phe as previously reported (45). The comparison of the NMR-fingerprints of the unmodified tRNA i Met and the m 5 C48,49-tRNA i Met therefore reveal that the introduction of m 5 Cs by Trm4 induces a certain stabilization in the folding of tRNA i Met , which could make it a better substrate for Trm6/Trm61 and explain the increased efficiency of m 1 A58 incorporation (Figure 3b, Tables 1 and 2).…”

“…To get a deeper understanding of the structural changes arising upon m 1 A58 introduction, we performed the assignment of the imino resonances of the m 1 A58-tRNA i Met following standard methods (Figure 4c), as previously described for other tRNAs (45). With this assignment at hand, we noticed that the imino signals of G18 and U55 are only visible in the spectrum of the m 1 A58-tRNA i Met (Figure 4a-c).…”

“…In both cases, the increased turnover rate might reflect a better substrate binding in a broad sense, which also means better substrate accommodation after a structural change via an induced-fit or conformational selection mechanism (62). The comparison of the NMR spectra of Ψ55- and T54-containing tRNA Phe with that of the unmodified tRNA Phe , showing very limited chemical shift variations, suggests that these modifications do not induce large global rearrangements in the structure of tRNA Phe (38,45). The effect of the modifications on the efficiency of the downstream enzymes is therefore most likely related to local and/or global changes in the dynamic properties of the tRNA substrate, as recently reported in the case of E. col i tRNAfMet, in which conformational fluctuations on the local level are increased in the modified tRNA (63).…”

A Method to Monitor the Introduction of Posttranscriptional Modifications in tRNAs with NMR Spectroscopy

“…One of the main challenge of NMR monitoring of tRNA maturation is to identify the NMR signature of individual modifications and thus to associate a particular chemical shift change on the NMR spectra with a specific tRNA modification event. For the yeast tRNA Phe , we assigned its imino groups in three forms differing in their modification content [24,27]. From the analysis of the differences between their ( 1 H, 15 N) chemical shifts, we could identify the NMR signature of individual modifications [15,27].…”

“…For the yeast tRNA Phe , we assigned its imino groups in three forms differing in their modification content [24,27]. From the analysis of the differences between their ( 1 H, 15 N) chemical shifts, we could identify the NMR signature of individual modifications [15,27]. The requirements for such a detailed analysis might be system dependent, but we anticipate that for any system, the complete NMR analysis of at least two tRNA samples, with and without modifications, would be crucial.…”