Pressimone Meghan A, Schissel Carly K, Goss Isabella H, Swenson Cameron V, Schepartz Alanna
Department of Molecular and Cellular Biology, University of California, Berkeley, CA 94720, United States.
Department of Chemistry, University of California, Berkeley CA 94720, United States.
Nucleic Acids Res. 2025 Apr 22;53(8). doi: 10.1093/nar/gkaf327.
We describe a new assay that reports directly on the acylation state of a user-chosen transfer RNA (tRNA) in cells. We call this assay 3-Prime Adenosine-Retaining Aminoacyl-tRNA Isolation (PARTI). It relies on high-resolution mass spectrometry identification of the acyl-adenosine species released upon RNase A cleavage of isolated cellular tRNA. Here we develop the PARTI workflow and apply it to understand three recent observations related to the cellular incorporation of non-α-amino acid monomers into protein: (i) the origins of the apparent selectivity of translation with respect to β2-hydroxy acid enantiomers; (ii) the activity of PylRS variants for benzyl derivatives of malonic acid; and (iii) the apparent inability of N-Me amino acids to function as ribosome substrates in living cells. Using the PARTI assay, we also provide direct evidence for the cellular production of 2',3'-diacylated tRNA in certain cases. The ease and simplicity of the PARTI workflow should benefit ongoing efforts to study and improve the cellular incorporation of non-α-amino acid monomers into proteins.
我们描述了一种新的检测方法，可直接报告细胞中用户选择的转运RNA（tRNA）的酰化状态。我们将此检测方法称为3'-腺苷保留氨酰-tRNA分离法（PARTI）。它依赖于通过高分辨率质谱法鉴定分离的细胞tRNA经核糖核酸酶A切割后释放的酰基腺苷种类。在此，我们开发了PARTI工作流程，并将其应用于理解与非α-氨基酸单体细胞内掺入蛋白质相关的三个最新观察结果：（i）翻译对β2-羟基酸对映体明显选择性的起源；（ii）吡咯赖氨酰-tRNA合成酶（PylRS）变体对丙二酸苄基衍生物的活性；（iii）N-甲基氨基酸在活细胞中作为核糖体底物的明显无功能状态。使用PARTI检测方法，我们还在某些情况下为细胞产生2'，3'-二酰化tRNA提供了直接证据。PARTI工作流程的简便性应有助于正在进行的研究和改进非α-氨基酸单体细胞内掺入蛋白质的工作。
