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

研究基因表达转录后调控过程中识别 RNA 甲基化的基本结构和序列特征

• 批准号: 10796260
• 负责人: Sanjaya Chinthaka Abeysirigunawardena
• 金额: $ 45.48万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10796260
• 关键词: Amino Acids; Binding; Binding Sites; Biological Assay; Biological Process; Cells; Code; Consensus; Consensus Sequence; Diabetes Mellitus; Dissociation; EMSA; Engineering; Equilibrium; Gene Expression Regulation; Genetic Transcription; Goals; Heterogeneous-Nuclear Ribonucleoproteins; Investigation; Kinetics; Link; Liquid substance; Male Infertility; Malignant Neoplasms; Messenger RNA; Methods; Methylation; Modification; Nucleotides; Obesity; Peptides; Phage Display; Phase; Play; Post-Transcriptional Regulation; Process; Protein Family; Proteins; Pseudouridine; RNA; RNA Binding; RNA Processing; RNA Sequences; RNA methylation; RNA-Protein Interaction; Reader; Reading; Regulation; Research; Role; Signal Transduction; Site; Sperm Maturation; Stress; Structure; Testing; Thermodynamics; Transcript; Translating; Tryptophan; Untranslated RNA; Virus Diseases; Work; base; epitranscriptomics; feature detection; hnRNP A1; hnRNP protein A1; human disease; nervous system disorder; novel; stopped-flow fluorescence; time use; transcriptome; tumor progression

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.

项目摘要 已知功能性RNA中存在RNA核苷酸修饰已有数十年。 最近的几项研究表明，转录组范围内存在核苷酸修饰， 如假尿苷、N6-甲基腺苷（m6 A）和5-甲基胞嘧啶。核苷酸水平 mRNA中的修饰处于紧密平衡，除非细胞处于各种应激条件下。 mRNA中m6 A水平的变化已显示影响病毒感染、精子成熟， 和癌症进展。在细胞中，m6 A水平由甲基写入器和读取器控制。这些 蛋白质将应激信号编码到mRNA转录物上，包括转录后和转录共。 识别甲基化的甲基阅读器在解码应激信号中起着关键作用， 引导mRNA进行编辑、加工、降解或翻译。考虑到更广泛的 在各种应激条件下和人类疾病中mRNA甲基化状态的多样性， 应该存在能够阅读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一致性（读者 或橡皮擦）阻碍读者发现新的调节机制，而橡皮擦不 已知最新。该项目的三个短期目标是1）发现序列或 与m6 A相互作用短肽的结构一致性，2）了解RNA结构如何 和序列可以改变m6 A-识别肽的序列和结构，3） 研究富集的肽抑制阅读器和擦除器蛋白的能力。 我们使用噬菌体展示方法来发现一个通用的序列或结构的共识， 识别核苷酸甲基化的蛋白质。我们建议测试RNA结构的影响 和富集肽的序列或结构上的序列。我们的下拉分析将 评价富集的肽模拟已知甲基读数的潜力。我们亦建议 为了比较针对甲基化靶标（噬菌体展示）和蛋白质选择的肽， 从pulldown测定中鉴定序列相似性。我们的初步工作表明：1）RNA 甲基化增强RNA序列特异性与蛋白质的相互作用，2）两个色氨酸 相隔4个氨基酸残基的残基可能在m6 A识别中发挥更大的作用3） 书写器或擦除器蛋白的RNA结合位点具有与所选肽相似的序列 分别针对未修饰的和修饰的RNA靶。我们的长期目标是 独特的设计蛋白质，其中m6 A识别肽（特异性结合序列 或结构特异性）与RNA加工、定位和 用于治疗人类疾病。