Berger Kyle D, Puthenpeedikakkal Anees M K, Mathews David H, Fu Dragony
Department of Biology, Center for RNA Biology, University of Rochester, Rochester, New York 14627, United States.
Department of Biochemistry & Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, New York 14642, United States.
bioRxiv. 2024 Nov 18:2024.11.18.624017. doi: 10.1101/2024.11.18.624017.
All tRNAs undergo a series of chemical modifications to fold and function correctly. In mammals, the C32 nucleotide in the anticodon loop of tRNA-Arg-CCU and UCU is methylated to form 3-methylcytosine (m3C). Deficiency of m3C in arginine tRNAs has been linked to human neurodevelopmental disorders, indicating a critical biological role for m3C modification. However, the structural repercussions of m3C modification are not well understood. Here, we examine the structural effects of m3C32 modification on the anticodon stem loop (ASL) of human tRNA-Arg-UCU-4-1, a unique tRNA with enriched expression in the central nervous system. Optical melting experiments demonstrate that m3C modification can locally disrupt nearby base pairing within the ASL while simultaneously stabilizing the ASL electrostatically, resulting in little net change thermodynamically. The isoenergetic nature of the C32 - A38 pair vs the m3C32 - A38 pair may help discriminate against structures not adopting canonical C32 - A38 pairings, as most other m3C pairings are unfavorable. Furthermore, multidimensional NMR reveals that after m3C modification there are changes in hairpin loop structure and dynamics, the structure of A37, and the neighboring A31 - U39 base pair. However, these structural changes after modification are made while maintaining the shape of the C32 - A38 pairing, which is essential for efficient tRNA function in translation. These findings suggests that m3C32 modification could alter interactions of tRNA-Arg isodecoders with one or more binding partners while simultaneously maintaining the tRNA's ability to function in translation.
所有转运RNA（tRNA）都要经历一系列化学修饰才能正确折叠并发挥功能。在哺乳动物中，tRNA-Arg-CCU和UCU反密码子环中的第32位核苷酸（C32）会发生甲基化，形成3-甲基胞嘧啶（m3C）。精氨酸tRNA中m3C的缺乏与人类神经发育障碍有关，这表明m3C修饰具有关键的生物学作用。然而，m3C修饰对结构的影响尚未得到充分理解。在这里，我们研究了m3C32修饰对人tRNA-Arg-UCU-4-1反密码子茎环（ASL）的结构影响，tRNA-Arg-UCU-4-1是一种在中枢神经系统中表达丰富的独特tRNA。光学熔解实验表明，m3C修饰可局部破坏ASL内附近的碱基配对，同时在静电作用下稳定ASL，导致热力学上净变化很小。C32 - A38碱基对与m3C32 - A38碱基对的等能性质可能有助于区分不采用经典C32 - A38配对的结构，因为大多数其他m3C配对是不利的。此外，多维核磁共振（NMR）显示，m3C修饰后发夹环结构和动力学、A37的结构以及相邻的A31 - U39碱基对都发生了变化。然而，修饰后的这些结构变化是在保持C32 - A38配对形状的同时发生的，这对于tRNA在翻译中高效发挥功能至关重要。这些发现表明，m3C32修饰可能会改变tRNA-Arg同功受体与一个或多个结合伙伴的相互作用，同时保持tRNA在翻译中发挥功能的能力。
