Investigation of the key structural and sequence features essential for m6A recognition during post-transcriphonal gene expression

项目总结 在功能RNA中存在RNA核苷酸修饰已经被知道了几十年。几个 最近的研究说明了转录组范围内核苷酸修饰的存在，例如伪尿嘧啶， N6-甲基腺苷(M6A)和5-甲基胞嘧啶。Mrna中的核苷酸修饰水平很低。 平衡，除非细胞处于不同的应力条件下。MRNAm6A水平的变化对病毒产生了影响 感染、精子成熟和癌症进展。在细胞中，m6A水平受甲基写入物和 读者。这些蛋白质将应激信号编码到转录后和共转录的mRNA转录本上。 识别甲基化的甲基阅读器在解码压力信号方面起着关键作用， 加工、降解或翻译信使核糖核酸。鉴于在不同的条件下，mRNA甲基化状态的多样性 在应激条件下和人类疾病中，一组甲基阅读器能够读取每个独特的 应存在应力信号。对甲基识别缺乏普遍的结构和序列共识 蛋白质(阅读器或擦除器)阻碍阅读器和擦除器发现新的调节机制 迄今为止已知的。该项目的三个短期目标是1)发现序列或结构上的共识 与m6A相互作用的短肽，2)了解RNA结构和序列如何改变 M6A识别多肽的序列和结构，3)研究富集肽对M6A的识别能力 抑制阅读器和橡皮擦蛋白。 我们使用噬菌体展示方法来发现蛋白质的一般序列或结构共识 识别核苷酸甲基化。我们建议测试RNA结构和序列对 富集肽的序列或结构。我们的下拉分析将评估富集物的潜力 模拟已知的甲基阅读器的多肽。我们还建议将选择的多肽与甲基化的多肽进行比较 目标(噬菌体展示)和从下拉分析中鉴定的蛋白质的序列相似性。我们的预赛 研究表明，1)RNA甲基化增强了RNA序列与蛋白质的特异性相互作用，2)2 色氨酸残基可能在m6A识别中发挥更重要的作用。 3)写入者或擦除者蛋白的rna结合位点与所选择的抗原肽具有相似的序列。 未修饰和修饰的RNA靶标。等温量热(ITC)是一种强有力的研究手段 生物分子之间的亲和力以及各种小分子配体之间的亲和力。我们建议使用ITC来衡量 多肽与各种修饰和未修饰的RNA寡核苷酸的结合亲和力。这些实验将允许 US以量化多肽与甲基化RNA寡核苷酸的结合特异性或偏好。此外，我们还将 使用ITC验证我们的RNA下拉检测中丰富的蛋白质。这项研究将有助于确定基本的 能够识别RNA中m6A甲基化的蛋白质/肽的序列特性。

PROJECT SUMMARY The presence of RNA nucleotide modifications in functional RNAs has been known for many decades. Several recent studies illustrate the transcriptome-wide presence of nucleotide modifications such as pseudouridines, N6-methyladenosines (m6A), and 5-methylcytosines. The levels of nucleotide modifications in mRNA are in tight equilibrium unless cells are under various stress conditions. Changes in m6A levels in mRNA have impacted viral infections, sperm maturation, and cancer progression. In cells, m6A levels are controlled by methyl writers and readers. These proteins codes the stress signal onto mRNA transcripts, both post- and co-transcriptionally. Methyl readers that recognize methylations play the critical role of decoding stress signals and either edit, process, degrade or translate mRNA. Given the broader diversity of mRNA methylation states under various stress conditions and in human diseases, an assemblage of methyl readers capable of reading each unique stress signal should exist. The lack of general structural and sequence consensus for methyl-recognizing proteins (reader or erasers) impedes the discovery of novel regulation mechanisms by readers and erasers not known to date. The three short term goals of this project are 1) to discover sequence or structural consensus for short peptides that interact with m6A, 2) to understand how RNA structure and sequence can change the sequence and the structure of m6A-recognizing peptides, 3) to investigate the ability of enriched peptides to inhibit reader and eraser protein. We used the phage display method to discover a general sequence or structural consensus for proteins recognizing nucleotide methylations. We propose to test the impact of RNA structure and sequence on the sequence or structure of the enriched peptides. Our pull-down assays will evaluate the potential of the enriched peptides to mimic known methyl readers. We also propose to compare the peptides selected against methylated targets (phage display) and proteins identified from pull-down assays for sequence similarity. Our preliminary work shows that 1) RNA methylations enhance the RNA sequence-specific interactions with proteins, 2) two tryptophan residues that reside four amino acid residues apart may play a more significant role in m6A recognition 3) RNA binding sites of writer or eraser proteins have similar sequences as the selected peptides against unmodified and modified RNA targets, respectively. Isothermal calorimetry (ITC) is a powerful technique to study the affinity of biomolecules to each other and various small molecular ligands. We propose using ITC to measure peptides' binding affinity to various modified and unmodified RNA oligonucleotides. These experiments will allow us to quantify peptides' binding specificity or preference to methylated RNA oligonucleotides. In addition, we will use ITC to validate protein enriched in our RNA pull-down assays. This research will help identify fundamental sequence properties of proteins/peptides that enable recognizing m6A methylations in RNA.