Investigation of the essential structural and sequence features for the recognition of RNA methylations during post-transcriptional regulation of gene expression

项目总结 几十年来，在功能RNA中存在RNA核苷酸修饰是已知的。 最近的几项研究表明，在转录组范围内存在核苷酸修饰，如 作为假尿苷、N6-甲基腺苷(M6A)和5-甲基胞嘧啶。核苷酸水平 除非细胞处于不同的压力条件下，否则mRNA的修饰处于紧平衡状态。 MRNA中m6A水平的变化已被证明影响病毒感染、精子成熟、 以及癌症的进展。在细胞中，m6A水平由甲基写入者和读取者控制。这些 蛋白质将压力信号编码到mRNA转录产物上，包括转录后和共转录。 识别甲基化的甲基阅读器在解码压力信号和 直接对信使基因进行编辑、处理、降解或翻译。考虑到更广泛的 不同应激条件下和人类疾病中mRNA甲基化状态的多样性 应该存在能够读取每个独特应力信号的甲基读取器的集合。 缺乏甲基识别蛋白的一般结构和序列共识(读者 或橡皮擦)阻碍了阅读器和橡皮擦新的调节机制的发现 直到现在才知道。该项目的三个短期目标是：1)发现序列或 与m6A相互作用的短肽的结构共识，2)了解RNA的结构 和序列可以改变m6A识别多肽的序列和结构，3) 研究富集肽对阅读器和橡皮擦蛋白的抑制能力。 我们使用噬菌体展示方法来发现一个普遍的序列或结构共识 识别核苷酸甲基化的蛋白质。我们建议测试RNA结构的影响 以及对富集肽的序列或结构进行测序。我们的下拉分析将会 评估富集肽模拟已知甲基阅读器的潜力。我们还提议 比较针对甲基化靶标(噬菌体展示)和蛋白质选择的多肽 从序列相似性的下拉分析中鉴定。我们的初步工作表明：1)RNA 甲基化增强RNA序列与蛋白质的特异性相互作用，2)两种色氨酸 相隔四个氨基酸残基的残基可能在m6A识别中发挥更大作用3) 编写器或擦除器蛋白的RNA结合位点与所选肽具有相似的序列 分别针对未修饰和修饰的RNA靶标。我们的长期目标是设计 独特的设计蛋白，其中M6A识别肽(特定的结合序列 或特定于结构)与与RNA加工、定位和 用于治疗人类疾病的降解物。

PROJECT SUMMARY The existence of RNA nucleotide modifications in functional RNAs is 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 been shown to impact viral infections, sperm maturation, and cancer progression. In cells, m6A levels are controlled by methyl writers and readers. These proteins code the stress signal on to mRNA transcripts, both post-, and co-transcriptionally. Methyl readers that recognize methylations play the critical role of decoding stress signals and direct mRNA to either getting edited, processed, degraded, or translated. Given the broader diversity of mRNA methylation states under various stress conditions and in human diseases, an assemblage of methyl readers that are 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 up 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 use phage display method to discover a general sequence or structural consensus for proteins that recognize nucleotide methylations. We propose to test the impact of RNA structure and sequence on the sequence or structure of the enriched peptides. Our pulldown 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 pulldown 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 greater 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. Our long-term objective is to engineer unique designer proteins in which m6A-recognizing peptides (that binding sequence specifically or structure specifically) are fused with proteins related to RNA processing, localization, and degradations to use in treating human diseases.