## 2 Influence of tRNA Core Modification on tRNA Structure and Stability
### Interdependence Involving tRNA Core Modifications
In particular, a strong modification circuit is present in the T-arm, in which the prior presence of V55 activates the formation of m\({}^{5}\)U54 by Trm2 and m\({}^{1}\)A58 by Trm6/Trm61, and m\({}^{5}\)U54 promotes the formation of m\({}^{1}\)A58 (Figure 3b). These cross-talks were shown to be also present in other yeast tRNAs, although they were not specifically identified [69]. A recent study conducted nanopore sequencing of all yeast tRNAs in specific deletion strains [112]. This study confirmed the modification circuit in the T-arm of tRNA\({}^{\text{The}}\) and further showed that it is also present in at least 15 other tRNA species [112]. Further biochemical studies on this V55 \(\rightarrow\) m\({}^{5}\)U54 \(\rightarrow\) m\({}^{1}\)A58 modification circuit, have characterized and quantified the direct effect of the initial modifications on the enzymatic activity of the subsequent ones [70]. The V55 and m\({}^{5}\)U54 modifications have a strong cumulative effect on the introduction of m\({}^{1}\)A58, together accelerating this process by a factor of \(\sim\)15 (discussed in [113]). Recently, several reports have shed light on modification cross-talks among tRNA core modifications in _E. coli_. They reported that the introduction of acp\({}^{3}\)U47 is globally  
Figure 3: Modification circuits involving tRNA core modifications: (**a**) modification circuits identified in _T. thermophilus_ tRNA\({}^{\text{Phe}}\) in response to temperature changes represented on a cloverleaf scheme [94; 95; 107]; (**b**) T-arm modification circuits identified in yeast tRNA\({}^{\text{Phe}}\)[69; 70]; (**c**,**d**) modification circuits identified in _E. coli_ tRNA\({}^{\text{Phe}}\)[109; 110; 111]; and (**e**) modification circuits identified in _E. coli_ tRNA\({}^{\text{Asp}}\)[111]. Modifications are represented by colored circles (see the legend in the box for the corresponding colors). Arrows indicate stimulatory effects between modifications, and blunted lines indicate inhibitory effects between modifications. enhanced by the prior presence of m\({}^{7}\)G46 [109] and also stimulated by m\({}^{5}\)U54 in _E. coli_ tRNA\({}^{\text{Arg}}\), \(t\)RNA\({}^{\text{Ile(GAU)}}\), \(t\)RNA\({}^{\text{Lys}}\), \(t\)RNA\({}^{\text{Phe}}\) and tRNA\({}^{\text{Val}}\)[110; 111] (Figure 3c). \(\Psi\)55 seems to also participate in this network, in which it may either slightly stimulate or inhibit the introduction of acp\({}^{3}\)U47 in _E. coli_ tRNA\({}^{\text{Phe}}\) and tRNA\({}^{\text{Lys}}\), respectively [111]. In addition, the formation of ms\({}^{2}\)!6A37 in the ACL of _E. coli_ tRNA\({}^{\text{Phe}}\) is activated by m\({}^{5}\)U54 [110; 111] (Figure 3d), and the introduction of s\({}^{4}\)U8 is enhanced in the presence of \(\Psi\)55 and m\({}^{5}\)U54 in _E. coli_ tRNA\({}^{\text{Asp}}\)[111] (Figure 3e). These interdependencies in _E. coli_ tRNA core modifications were identified using a combination of nucleotide quantification and sequencing techniques, such as HPLC, liquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS), multiplex small RNA-sequencing (MSR-seq) and primer extension analysis. Finally, an order for the introduction of modifications into _E. coli_ tRNA\({}^{\text{Phe}}\) was suggested where \(\Psi\)55 and m\({}^{5}\)U54 are introduced in the earliest stages of maturation, followed by m\({}^{7}\)G46 and then acp\({}^{3}\)U47, with s\({}^{4}\)U8 being introduced after \(\Psi\)55 and m\({}^{5}\)U54 [114]. Apart from the few examples described above, the characterization of these interdependencies is still relatively limited, but the latest technological developments, in particular in nanopore sequencing technology, are expected to accelerate the discovery of modification circuits involving tRNA core modifications [115].