Eukaryotic AlaX provides multiple checkpoints for quality and quantity of aminoacyl-tRNAs in translation.

Translational fidelity relies critically on correct aminoacyl-tRNA supply. The trans-editing factor AlaX predominantly hydrolyzes Ser-tRNAAla, functioning as a third sieve of alanyl-tRNA synthetase (AlaRS). Despite extensive studies in bacteria and archaea, the mechanism of trans-editing in mammals remains largely unknown. Here, we show that human AlaX (hAlaX), which is exclusively distributed in the cytoplasm, is an active trans-editing factor with strict Ser-specificity. In vitro, both hAlaX and yeast AlaX (ScAlaX) were capable of hydrolyzing nearly all Ser-mischarged cytoplasmic and mitochondrial tRNAs; and robustly edited cognate Ser-charged cytoplasmic and mitochondrial tRNASers. In vivo or cell-based studies revealed that loss of ScAlaX or hAlaX readily induced Ala- and Thr-to-Ser misincorporation. Overexpression of hAlaX impeded the decoding efficiency of consecutive Ser codons, implying its regulatory role in Ser codon decoding. Remarkably, yeast cells with ScAlaX deletion responded differently to translation inhibitor treatment, with a gain in geneticin resistance, but sensitivity to cycloheximide, both of which were rescued by editing-capable ScAlaX, alanyl- or threonyl-tRNA synthetase. Altogether, our results demonstrated the previously undescribed editing peculiarities of eukaryotic AlaXs, which provide multiple checkpoints to maintain the speed and fidelity of genetic decoding.

The alanyl-tRNA synthetase AARS1 moonlights as a lactyltransferase to promote YAP signaling in gastric cancer.

Lactylation has been recently identified as a new type of posttranslational modification occurring widely on lysine residues of both histone and nonhistone proteins. The acetyltransferase p300 is thought to mediate protein lactylation, yet the cellular concentration of the proposed lactyl-donor, lactyl-coenzyme A, is about 1,000 times lower than that of acetyl-CoA, raising the question of whether p300 is a genuine lactyltransferase. Here, we report that alanyl-tRNA synthetase 1 (AARS1) moonlights as a bona fide lactyltransferase that directly uses lactate and ATP to catalyze protein lactylation. Among the candidate substrates, we focused on the Hippo pathway, which has a well-established role in tumorigenesis. Specifically, AARS1 was found to sense intracellular lactate and translocate into the nucleus to lactylate and activate the YAP-TEAD complex; and AARS1 itself was identified as a Hippo target gene that forms a positive-feedback loop with YAP-TEAD to promote gastric cancer (GC) cell proliferation. Consistently, the expression of AARS1 was found to be upregulated in GC, and elevated AARS1 expression was found to be associated with poor prognosis for patients with GC. Collectively, this work found AARS1 with lactyltransferase activity in vitro and in vivo and revealed how the metabolite lactate is translated into a signal of cell proliferation.

Elucidation of productive alanine recognition mechanism by Escherichia coli alanyl-tRNA synthetase.

Alanyl-tRNA synthetase (AlaRS) incorrectly recognizes both a slightly smaller glycine and a slightly larger serine in addition to alanine, and the probability of incorrect identification is extremely low at 1/300 and 1/170, respectively. Alanine is the second smallest amino acid after glycine; however, the mechanism by which AlaRS specifically identifies small differences in side chains with high accuracy remains unknown. In this study, using a malachite green assay, we aimed to elucidate the alanine recognition mechanism of a fragment (AlaRS368N) containing only the amino acid activation domain of Escherichia coli AlaRS. This method quantifies monophosphate by decomposing pyrophosphate generated during aminoacyl-AMP production. AlaRS368N produced far more pyrophosphate when glycine or serine was used as a substrate than when alanine was used. Among several mutants tested, an AlaRS mutant in which the widely conserved aspartic acid at the 235th position (D235) near the active center was replaced with glutamic acid (D235E) increased pyrophosphate release for the alanine substrate, compared to that from glycine and serine. These results suggested that D235 is optimal for AlaRS to specifically recognize alanine. Alanylation activities of an RNA minihelix by the mutants of valine at the 214th position (V214) of another fragment (AlaRS442N), which is the smallest AlaRS with alanine charging activity, suggest the existence of the van der Waals-like interaction between the side chain of V214 and the methyl group of the alanine substrate.