ABSTRACT

RNA modifications are dynamic chemical entities that expand the RNA lexicon and regulate RNA fate. The most abundant modification present in mRNAs, N6-methyladenosine (m⁶A), has been implicated in neurogenesis and memory formation. However, whether additional RNA modifications may be playing a role in neuronal functions and in response to environmental queues is largely unknown. Here we characterize the biochemical function and cellular dynamics of two human RNA methyltransferases previously associated with neurological dysfunction, TRMT1 and its homolog, TRMT1-like (TRMT1L). Using a combination of next-generation sequencing, LC-MS/MS, patient-derived cell lines and knockout mouse models, we confirm the previously reported dimethylguanosine (m^2,2G) activity of TRMT1 in tRNAs, as well as reveal that TRMT1L, whose activity was unknown, is responsible for methylating a subset of cytosolic tRNA^Ala(AGC) isodecoders at position 26. Using a cellular in vitro model that mimics neuronal activation and long term potentiation, we find that both TRMT1 and TRMT1L change their subcellular localization upon neuronal activation. Specifically, we observe a major subcellular relocalization from mitochondria and other cytoplasmic domains (TRMT1) and nucleoli (TRMT1L) to different small punctate compartments in the nucleus, which are as yet uncharacterized. This phenomenon does not occur upon heat shock, suggesting that the relocalization of TRMT1 and TRMT1L is not a general reaction to stress, but rather a specific response to neuronal activation. Our results suggest that subcellular relocalization of RNA modification enzymes may play a role in neuronal plasticity and transmission of information, presumably by addressing new targets.

摘要

RNA 修饰是动态的化学实体，可扩展 RNA 词典并调节 RNA 命运。mRNA 中最丰富的修饰，N6-甲基腺苷 (m ⁶ A)，与神经发生和记忆形成有关。然而，额外的 RNA 修饰是否可能在神经元功能和响应环境队列中发挥作用在很大程度上是未知的。在这里，我们描述了先前与神经功能障碍相关的两种人类 RNA 甲基转移酶 TRMT1 及其同源物 TRMT1样(TRMT1L) 的生化功能和细胞动力学。结合新一代测序、LC-MS/MS、患者来源的细胞系和基因敲除小鼠模型，我们证实了先前报道的二甲基鸟苷（m ^2,2G) TRMT1 在 tRNA 中的活性，并揭示了活性未知的TRMT1L负责甲基化第 26 位胞质 tRNA ^Ala (AGC) 同工解码器的子集。在模拟神经元激活和长期增强的模型中，我们发现 TRMT1 和 TRMT1L 在神经元激活时都改变了它们的亚细胞定位。具体来说，我们观察到从线粒体和其他细胞质结构域 (TRMT1) 和核仁 (TRMT1L) 到细胞核中不同的小点状区室的主要亚细胞重新定位，这尚未被表征。这种现象在热休克时不会发生，这表明 TRMT1 和 TRMT1L 的重新定位不是对压力的一般反应，而是对神经元激活的特定反应。我们的研究结果表明，RNA 修饰酶的亚细胞重定位可能在神经元可塑性和信息传递中发挥作用，可能是通过解决新目标。