人源ADAR1蛋白RNA编辑机制的研究

RNA编辑是一种重要的转录后修饰方式，经过RNA编辑，其编码区发生碱基的加入、丢失及转换现象，最终导致遗传信息的改变，达到其校正、翻译调控以及扩充遗传信息等作用。ADAR1作为主要的RNA编辑酶，介导双链RNA底物分子上的腺嘌呤脱氨基成为次黄嘌呤（A-to-I），翻译机器将I（肌苷）读作G（鸟嘌呤），以达到改变蛋白序列或调控蛋白质表达的作用。ADAR1自作为RNA解旋酶被发现以来，其功能得到了广泛的研究，特别是作为免疫调节因子以及其对癌症发生发展的作用机制。然而由于缺乏ADAR1蛋白的三维结构信息，ADAR1蛋白的RNA编辑分子机制目前还尚未明确。因此，阐明ADAR1蛋白的RNA编辑分子机制、活性调控机制是本研究的目的。本研究尝试利用冷冻电子显微镜来解析ADAR1的全长结构并从结构上探索其RNA编辑机制，由于全长的ADAR1蛋白极易降解的性质以及其N端大量的柔性结构，解析ADAR1蛋白全长以及其截短体（3/2/1个dsRNA结合结构域+脱氨酶结构域）的结构具有很大的挑战性。我们发现在细胞内过表达ADAR1的N端第1-125以及第1-490个氨基酸时，能够观察到相分离现象，在体外第1-125个氨基酸在有核酸底物加入的情况下也能出现相分离，而第1-490个氨基酸片段则受制于低表达和低纯度，暂时无法在体外观察到此现象，我们推测ADAR1液液相分离的性质与Zα DNA结合结构域密切相关。我们研究了双链RNA结合结构域对ADAR1编辑活性的影响，并且发现了RNA解旋酶ZNFX1与ADAR1相互作用并且能够影响ADAR1的脱氨酶活性。本研究为继续解析ADAR1全长结构提供了思路和方法，鉴定出解旋酶ZNFX1与ADAR1蛋白存在直接相互作用，并调控ADAR1蛋白的RNA编辑活性，一定程度上解释了ADAR1抑制应激颗粒形成的机制，为后续的深入研究ADAR1蛋白的RNA编辑机制及活性调控机制奠定了扎实的基础。

RNA editing is an important post-transcriptional modification pattern. After RNA editing, the insertion, deletion and conversion of bases in the coding region will eventually lead to the change of genetic information, and achieve its functions such as replicational error correction, translation regulation and expansion of genetic information. ADAR1, as the main RNA editing enzyme, mediates the deamination of adenine on dsRNA and converts adenosine (A) to inosine (I), and the translation machine reads I (inosine) as G (guanine) to achieve the effect of changing protein sequence or regulate protein expression. Since its discovery as an RNA helicase, functions of ADAR1 have been extensively studied, especially as an immune regulator and its role in the development of cancer. However, due to the lack of 3D structural information of ADAR1 protein, the mechanism of RNA editing of ADAR1 protein has not yet been elucidated. Therefore, the purpose of the current study is to elucidate the RNA editing molecular mechanism and activity regulation mechanism of ADAR1. This study attempts to use cryo-EM to analyze the full-length and truncated structure of ADAR1. Due to the highly degradable nature of full-length ADAR1 protein and the large number of flexible structures at its N-terminus fragment (1-490aa), it is challenging to analyze the structure of full-length ADAR1 and its truncations. Besides, liquid-liquid phase separation could be observed when the N-terminal amino acids 1-125 (ADAR1-p150(1-125)) and 1-490 (ADAR1-p150(1-490)) of ADAR1 were overexpressed in cells. The phase separation of ADAR1-p150(1-125) was observed in the presence of total RNA in vitro. While the ADAR1-p150(1-490) is subject to low expression and purity, and this phenomenon of LLPS cannot be observed in vitro. We speculate that the LLPS property of ADAR1 is closely related to the Z𝛼 domain. we investigated the effect of the dsRBDs on ADAR1 editing activity and found that the RNA helicase ZNFX1 interacts with ADAR1 which could affect ADAR1 deaminase activity. This study provides ideas and methods for continuing to analyze the full-length structure of ADAR1 and identifies the direct interaction between ZNFX1 and ADAR1 protein which could regulate the RNA editing activity of ADAR1 protein. The mechanism laid a solid foundation for the subsequent in-depth study of the RNA editing mechanism and activity regulation mechanism of ADAR1 protein.

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