tRNAArg-Derived Fragments Can Serve as Arginine Donors for Protein Arginylation

Hijacking tRNAs From Translation: Regulatory Functions of tRNAs in Mammalian Cell Physiology

“…In fact, it is a general question for all of the processes which rely on the use of charged tRNAs. Although it remains largely elusive, tRNA fragmentation may serve a mechanism that potentially shifts the balance depending on the demand of the cell for either translation or arginylation (Avcilar-Kucukgoze et al, 2020). On the other hand, tRNAs with specific posttranscriptional modifications may be dedicated to particular non-translational processes.…”

“…Our recent study showed that arginyl-tRF Arg can be generated in vitro from Arg-tRNA Arg using RNase T2 and that such Arg-tRF Arg can mediate arginylation (Avcilar-Kucukgoze et al, 2020; Figure 5). As translation and arginylation compete for the same substrate, arginyl-tRNA Arg , such aminoacyl-tRF generation in this case, if it happens in vivo, may serve as a switch between these two processes (Avcilar-Kucukgoze et al, 2020). In support, lack of Ate1 significantly alters the ratio of tRNA Arg :tRF Arg in MEFs, suggesting a functional link between tRF Arg and arginylation in vivo (Avcilar-Kucukgoze et al, 2020).…”

“…As translation and arginylation compete for the same substrate, arginyl-tRNA Arg , such aminoacyl-tRF generation in this case, if it happens in vivo, may serve as a switch between these two processes (Avcilar-Kucukgoze et al, 2020). In support, lack of Ate1 significantly alters the ratio of tRNA Arg :tRF Arg in MEFs, suggesting a functional link between tRF Arg and arginylation in vivo (Avcilar-Kucukgoze et al, 2020). We propose that arginyl-tRF Arg generation is a mechanism that maintains the balance between translation and arginylation and can potentially shift this balance in favor of one or the other process in response to physiological stimuli.…”

A Label-Free Assay for Aminoacylation of tRNA

“…We chose E. coli tRNA Ser/GCU from the collection above as the substrate ( Figure 2 b), which was readily available in large quantities in the purified transcript form. As a proof of principle, we selected the following amino acid analogs ( Figure 3 a): cis -hydroxyproline, trans -hydroxyproline, azetidine-2-carboxylic acid (abbreviated as azetidine, an analog of proline), citrulline (a precursor of arginine), and N-ethylmaleimide (NEM)-cysteine (where the NEM group is attached to the sulfur of cysteine), each of which was previously chemically synthesized as a DBE-conjugate and was readily trans-esterified to tRNA by dFx flexizyme [ 6 , 45 ]. Aminoacylation of E. coli tRNA Ser/GCU with each analog, followed by biotin-SA conjugation and analysis by a denaturing/7 M urea gel, showed that all of the non-proteinogenic aa-tRNA products were captured by biotin-SA conjugation, resulting in upshifted bands, whereas the control reaction lacking an aa-DBE had no shift ( Figure 3 b).…”

“…Additionally, some aa-tRNAs are associated with non-ribosomal cellular activities. A well-known example of the latter is post-translational arginylation to protein catalyzed by arginyl transferases, which use Arg-tRNA Arg as the aminoacyl donor for transferring to protein substrates as the marker for degradation [ 5 , 6 ]. In both ribosome-dependent and ribosome-independent activities, the level of each aminoacylation reaction determines the flow and the amount of the aa-tRNA that supports these activities.…”