Faculty of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
Genes (Basel). 2023 Feb 10;14(2):462. doi: 10.3390/genes14020462.
Aminoacyl tRNA synthetases (aaRSs) are a well-studied family of enzymes with a canonical role in charging tRNAs with a specific amino acid. These proteins appear to also have non-canonical roles, including post-transcriptional regulation of mRNA expression. Many aaRSs were found to bind mRNAs and regulate their translation into proteins. However, the mRNA targets, mechanism of interaction, and regulatory consequences of this binding are not fully resolved. Here, we focused on yeast cytosolic threonine tRNA synthetase (ThrRS) to decipher its impact on mRNA binding. Affinity purification of ThrRS with its associated mRNAs followed by transcriptome analysis revealed a preference for mRNAs encoding RNA polymerase subunits. An mRNA that was significantly bound compared to all others was the mRNA encoding RPC10, a small subunit of RNA polymerase III. Structural modeling suggested that this mRNA includes a stem-loop element that is similar to the anti-codon stem loop (ASL) structure of ThrRS cognate tRNA (tRNA). We introduced random mutations within this element and found that almost every change from the normal sequence leads to reduced binding by ThrRS. Furthermore, point mutations at six key positions that abolish the predicted ASL-like structure showed a significant decrease in ThrRS binding with a decrease in RPC10 protein levels. Concomitantly, tRNA levels were reduced in the mutated strain. These data suggest a novel regulatory mechanism in which cellular tRNA levels are regulated through a mimicking element within an RNA polymerase III subunit in a manner that involves the tRNA cognate aaRS.
氨酰-tRNA 合成酶（aaRSs）是一类研究较为透彻的酶家族，其在将特定氨基酸加载到 tRNA 上具有典型作用。这些蛋白质似乎还具有非典型作用，包括对 mRNA 表达的转录后调控。许多 aaRS 被发现与 mRNA 结合并调节其翻译成蛋白质。然而，这种结合的 mRNA 靶标、相互作用机制和调节后果尚未完全解决。在这里，我们专注于酵母细胞质中的苏氨酸 tRNA 合成酶（ThrRS），以阐明其对 mRNA 结合的影响。用其相关的 mRNA 亲和纯化 ThrRS 后进行转录组分析，揭示了对编码 RNA 聚合酶亚基的 mRNA 的偏好。与所有其他 mRNA 相比，显著结合的 mRNA 是编码 RNA 聚合酶 III 小亚基 RPC10 的 mRNA。结构建模表明，该 mRNA 包含一个茎环元件，类似于 ThrRS 同源 tRNA（tRNA）的反密码子茎环（ASL）结构。我们在该元件内引入随机突变，发现几乎所有正常序列的改变都会导致 ThrRS 结合减少。此外，在六个关键位置的点突变使预测的 ASL 样结构被破坏，导致 ThrRS 结合显著减少，RPC10 蛋白水平降低。同时，突变株中的 tRNA 水平降低。这些数据表明了一种新的调节机制，其中细胞内 tRNA 水平通过 RNA 聚合酶 III 亚基内的模拟元件进行调节，这种调节方式涉及到 tRNA 同源 aaRS。
