Distinct small non-coding RNA landscape in the axons and released extracellular vesicles of developing primary cortical neurons and the axoplasm of adult nerves

Neurons have highlighted the needs for decentralized gene expression and specific RNA function in somato-dendritic and axonal compartments, as well as in intercellular communication via extracellular vesicles (EVs). Despite advances in miRNA biology, the identity and regulatory capacity of other small non-coding RNAs (sncRNAs) in neuronal models and local subdomains has been largely unexplored. We identified a highly complex and differentially localized content of sncRNAs in axons and EVs during early neuronal development of cortical primary neurons and in adult axons <i>in</i> <i>vivo</i>. This content goes far beyond miRNAs and includes most known sncRNAs and precisely processed fragments from tRNAs, sno/snRNAs, Y RNAs and vtRNAs. Although miRNAs are the major sncRNA biotype in whole-cell samples, their relative abundance is significantly decreased in axons and neuronal EVs, where specific tRNA fragments (tRFs and tRHs/tiRNAs) mainly derived from tRNAs Gly-GCC, Val-CAC and Val-AAC predominate. Notably, although 5ʹ-tRHs compose the great majority of tRNA-derived fragments observed <i>in</i> <i>vitro</i>, a shift to 3ʹ-tRNAs is observed in mature axons <i>in</i> <i>vivo</i>. The existence of these complex sncRNA populations that are specific to distinct neuronal subdomains and selectively incorporated into EVs, equip neurons with key molecular tools for spatiotemporal functional control and cell-to-cell communication.

神经元研究已凸显出，在胞体-树突与轴突区域，以及通过细胞外囊泡（extracellular vesicles, EVs）进行的细胞间通讯中，去中心化基因表达与特定RNA功能的必要性。尽管微小RNA（miRNA）生物学研究已取得诸多进展，但其余小型非编码RNA（small non-coding RNAs, sncRNAs）在神经元模型及其局部亚结构域中的身份与调控能力，在很大程度上仍未得到系统探索。本研究在皮层原代神经元的早期发育阶段以及体内成熟轴突中，鉴定出轴突与细胞外囊泡内存在高度复杂且定位特征各异的sncRNAs组分。该组分的涵盖范围远不止微小RNA，还包含绝大多数已报道的sncRNAs，以及来自转运RNA（tRNAs）、核仁小RNA/核小RNA（sno/snRNAs）、Y RNA与穹隆RNA（vtRNAs）的精准加工片段。尽管微小RNA是全细胞样本中的主要sncRNA生物类型，但它们在轴突与神经元细胞外囊泡中的相对丰度显著降低；此类样本中占主导的是主要源自甘氨酸-tRNA（Gly-GCC）、缬氨酸-tRNA（Val-CAC）与缬氨酸-tRNA（Val-AAC）的特定tRNA片段（tRFs与tRHs/tiRNAs）。值得注意的是，尽管体外实验中观测到的tRNA衍生片段绝大多数为5'-tRHs，但在体内成熟轴突中则呈现出向3'-tRNA片段转变的特征。这些特异性分布于不同神经元亚结构域、并被选择性包裹入细胞外囊泡的复杂sncRNA群体，为神经元提供了实现时空功能调控与细胞间通讯的关键分子工具。