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

Abstract

Arginyltransferase ATE1 mediates posttranslational arginylation and plays key roles in multiple physiological processes. ATE1 utilizes arginyl (Arg)-tRNA^Arg as the donor of Arg, putting this reaction into a direct competition with the protein synthesis machinery. Here, we address the question of ATE1- Arg-tRNA^Arg specificity as a potential mechanism enabling this competition in vivo. Using in vitro arginylation assays and Ate1 knockout models, we find that, in addition to full-length tRNA, ATE1 is also able to utilize short tRNA^Arg fragments that bear structural resemblance to tRNA-derived fragments (tRF), a recently discovered class of small regulatory non-coding RNAs with global emerging biological role. Ate1 knockout cells show a decrease in tRF^Arg generation and a significant increase in the ratio of tRNA^Arg:tRF^Arg compared with wild type, suggesting a functional link between tRF^Arg and arginylation. We propose that generation of physiologically important tRFs can serve as a switch between translation and protein arginylation.

Keywords: arginylation; tRF; tRNA.

Figure 1.. ATE1-mediated arginylation is highly specific toward Arg-tRNA^Arg.

MALDI-TOF chromatograms of angiotensin II. Masses of unmodified and arginylated angiotensin are marked. Only tRNA^Arg charged with Arg is able to mediate arginylation.

Figure 2.. ATE1 selectively binds to tRNA.

A, Gels of total RNA used as pulldown input, flow through (FT) and RNA eluate from His-tagged ATE1 immobilized on Ni-NTA beads (top), His-tagged gelsolin fragment immobilized on Ni-NTA beads (middle) and Ni-NTA beads alone (bottom). B, Comparative microarrays of tRNAs bound to ATE1 vs total tRNA of mouse liver summary of three biological replicates. Arrays are highly reproducible (Fig. S2). tRNA probes are depicted with their codon and the corresponding amino acid; Meti-ATG, initiator tRNA^Met. Two different probes recognizing different tRNA^Leu (Leu-UAA) and tRNA^Lys (Lys-UUU) isodecoders that pair to the same codon TTA/G-Leu or AAA-Lys codon but differ in their sequence outside the anticodon were used on the arrays. Data are shown as fold enrichment (gradient ruler at the bottom). Significantly enriched tRNAs in the ATE1 pull down compared to the total liver tRNAs were selected based on the confidence intervals of covariance analysis (an example between replicate 1 and 2 is included). C, Eluted RNA sample in denaturing 10% polyacrylamide gel. M, RNA marker.

Figure 3.. ATE1 is able to utilize all mouse tRNA^Arg species.

A, Rate of the arginylation reaction as a function of E. coli Arg-tRNA^Arg in the presence of excess of angiotensin II substrate B, Normalized Arg incorporation into angiotensin II using various pre-charged mouse Arg-tRNA^Arg isoacceptors and isodecoders. No tRNA, control without adding tRNA (see Table S1 for tRNA gene names and sequences, corresponding to the numbers on the chart).

Figure 4.. tRNA^Arg acceptor stem-like structures charged with Arg can mediate arginylation.

MALDI-TOF chromatograms of angiotensin II, arginylated in vitro using flexizyme-charged Arg-mini- and microhelix (left and middle panel), or RARS-charged Arg-tRNA^Arg. Masses of unmodified and arginylated angiotensin are marked. Structures of micro- and minihelix tRNA^Arg used in the reaction are shown on each chromatogram. The higher arginylated peak intensity in the tRNA-mediated reaction is likely due to the fact that this is a fully enzymatic reaction, where RARS present in the mixture continually charges tRNA for the arginylation reaction. Notably, the peak height/intensity in this method does not directly reflect the abundance of the peptide (Szajli et al., 2008).

Figure 5.. tRNA^Arg-derived fragments mediate arginylation.

A, tRNA cleavage pattens by Dicer and RNase T2. The arrows show the cleavage products. B, Autoradiogram of ¹⁴C-Arg-tRNA^Arg cleaved with RNase T2. C, normalized incorporation of ³H-Arg into angiotensin II performed with ³H-Arg-tRNA^Arg or ³H-Arg-tRF^Arg. Data are mean ± SEM of three biological replicates. D. MALDI-TOF chromatogram of angiotensin II arginylated using purified pre-charged ¹³C¹⁵N-Arg-tRNA^Arg or ¹³C¹⁵N-Arg-tRF^Arg. Notably, the peak height/intensity in this method does not directly reflect the abundance of the peptide (Szajli et al., 2008).

Figure 6.. ATE1 deletion alters the ratio of tRNA^Arg to tRF^Arg in cells.

A. Ratios of tRNA^Arg/tRF^Arg in the total RNA preparations from wild type (WT) and ATE1 knockout (ATE1 KO) mouse embryonic fibroblasts, analyzed in 3 biological replicates. Error bars represent SEM, Welch’s one-tailed T-test was used for calculating statistical significance. B, Hypothetical model of the arginylation and translation dependent tRNA^Arg/tRF^Arg balance.