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

研究转录后基因表达过程中 m6A 识别所必需的关键结构和序列特征

• 批准号: 10582122
• 负责人: Sanjaya Chinthaka Abeysirigunawardena
• 金额: $ 10万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2023-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10582122
• 关键词: Affect; Affinity; Amino Acids; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biophysics; Calorimetry; Cells; Circular Dichroism; Code; Consensus; Consensus Sequence; Equilibrium; Fluorescence; Fluorescence Resonance Energy Transfer; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; Growth; Investigation; Ligands; Malignant Neoplasms; Measurement; Measures; Messenger RNA; Methods; Methylation; Modification; Molecular; Nucleotides; Oligonucleotides; Peptides; Phage Display; Play; Process; Property; Proteins; Pseudouridine; RNA; RNA Binding; RNA Folding; RNA Sequences; RNA methylation; RNA-Protein Interaction; Reader; Reading; Regulation; Research; Role; Signal Transduction; Specificity; Spectrum Analysis; Sperm Maturation; Stress; Structure; Techniques; Testing; Transcript; Translating; Tryptophan; Untranslated RNA; Virus Diseases; Work; base; epitranscriptomics; experimental study; human disease; melting; novel; preference; protein aminoacid sequence; transcriptome; tumor progression

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.

项目摘要 在功能性RNA中RNA核苷酸修饰的存在已经被知道了几十年。几 最近的研究说明了核苷酸修饰如假尿苷的转录组范围的存在， N6-甲基腺苷（m6 A）和5-甲基胞嘧啶。mRNA中的核苷酸修饰水平与基因组中的核苷酸修饰水平密切相关。 除非细胞处于各种应力条件下。mRNA中m6 A水平的变化影响了病毒 感染、精子成熟和癌症进展。在细胞中，m6 A水平由甲基书写器控制， 读者这些蛋白质将应激信号编码到mRNA转录物上，包括转录后和共转录。 识别甲基化的甲基阅读器在解码应激信号中起着关键作用， 处理、降解或翻译mRNA。考虑到在各种条件下mRNA甲基化状态的更广泛多样性， 压力条件下和人类疾病中，能够阅读每个独特的甲基阅读器的集合 压力信号应该存在。缺乏甲基识别的一般结构和序列一致性 蛋白质（阅读器或橡皮擦）阻碍阅读器和橡皮擦发现新的调节机制， 已知日期。该项目的三个短期目标是：1）发现序列或结构一致性， 与m6 A相互作用的短肽，2）了解RNA结构和序列如何改变 序列和m6 A-识别肽的结构，3）研究富集肽的能力， 抑制阅读器和擦除器蛋白。 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一致性 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的 肽来模拟已知的甲基阅读器。我们还建议将选择的肽与甲基化的肽进行比较， 靶（噬菌体展示）和从下拉测定法鉴定的蛋白质的序列相似性。我们的初步 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）两种 相隔4个氨基酸残基的色氨酸残基可能在m6 A识别中起更重要的作用 3)书写器或擦除器蛋白质的RNA结合位点具有与所选择的针对 未修饰的和修饰的RNA靶标。等温量热法（ITC）是一种强大的技术， 生物分子彼此之间以及与各种小分子配体之间的亲和力。我们建议使用ITC来衡量 肽对各种修饰的和未修饰的RNA寡核苷酸的结合亲和力。这些实验将使 我们定量肽的结合特异性或偏好甲基化的RNA寡核苷酸。此外，我们将 使用ITC来验证在我们的RNA下拉分析中富集的蛋白质。这项研究将有助于确定基本的 能够识别RNA中m6 A甲基化的蛋白质/肽的序列性质。