A Non-Canonical RNA Structure Regulates Pre-miRNA Maturation

非规范 RNA 结构调节 Pre-miRNA 成熟

• 批准号: 8958542
• 负责人: Soumitra Basu
• 金额: $ 37.47万
• 依托单位: KENT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-07 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8958542
• 关键词: A549; Adopted; Affect; Binding; Biochemical; Biogenesis; Bioinformatics; Biological Models; Brain Neoplasms; Cells; Cellular biology; Cleaved cell; Code; Cytoplasm; Data; Databases; Development; Dicer Enzyme; Dimensions; Disease; Double-Stranded RNA; Drug resistance; Future; G-Quartets; Gene Expression; Gene Targeting; Genes; Glioblastoma; Goals; Housing; Human; Immune; Immune System Diseases; In Vitro; Lead; Link; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; MicroRNAs; Molecular; Molecular Conformation; Nerve Degeneration; Non-Small-Cell Lung Carcinoma; Oligonucleotides; PTEN gene; Play; Primary Brain Neoplasms; Process; Production; Proteins; RNA; RNA Sequences; Regulation; Reporting; Ribonucleases; Role; Stretching; Structure; Testing; Therapeutic; Vertebrates; Virus Diseases; biophysical techniques; cancer cell; human DICER1 protein; human FBXW7 protein; human disease; novel therapeutics; overexpression; public health relevance; small molecule; stem; tool; transcriptome sequencing

 DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are short oligonucleotides that regulate more than one half of protein coding genes' expression in human. MicroRNAs have been associated with common human diseases such cancer, immune disorders, viral infections and neurodegeneration. Since elevated miRNA levels are often linked to various disease states, the biogenesis of miRNA is a tightly regulated process. Dicer mediated maturation of precursor miRNA (pre-miRNA) in the cytoplasm is a critical step in miRNA biogenesis where RNase enzyme Dicer recognizes and internally cleaves the pre-miRNA stem-loop to produce the mature miRNA. The current dogma is that all pre-miRNAs adopt a stem-loop structure, which is required for the accurate maturation by Dicer. Thus, besides the canonical stem-loop structure, no other alternative secondary structures have been actively considered for the pre-miRNAs. However, after comprehensive analysis of all known human pre-miRNA and mature miRNA sequences, we identified 298 and 179 sequences respectively which have the propensity to form a stable G-quadruplex (GQ) as an alternative RNA secondary structure. In this proposal, we intend to investigate in detail the role of GQ structure in miRNA biogenesis and the ability of small molecules to control the pre-miRNA maturation by targeting the GQ structures. Using a clinically important miRNA (pre-miRNA 92b) which is overexpressed in various diseases, we showed the structural coexistence of GQ and the canonical stem-loop structures of pre-miRNA under the physiologically relevant K+ concentration. Additionally, our preliminary data show that the GQ formation inhibits the Dicer mediated maturation of human pre-miRNA 92b. Given these findings, we hypothesize that GQ structures play a crucial role in miRNA biogenesis at both pre-miRNA maturation and mature duplex miRNA strand separation. We also hypothesize that small molecule binding to the GQs in pre-miRNA can modulate its maturation. These hypotheses will be investigated under the specific aims: (I) to determine the effect of the GQ on Dicer mediated pre-miRNA maturation using human pre-miRNA 92b, (II) to delineate whether the GQ formation in the mature miRNAs causes the structural unwinding of the miRNA duplex; and (III) to determine if small molecule targeted to non-canonical structure in pre-miRNA can modulate its maturation by Dicer in vitro and in human cells. Completion of these specific aims will reveal the importance of alternative secondary structures in miRNA biogenesis, which in turn will lead to better understanding of how miRNA levels control various diseases. The targeting of GQs with small molecule will be a new way to control the endogenous miRNA biogenesis, which can be used for development of novel therapeutics. This comprehensive study will not only add a new dimension to our current understanding of the RNA structural role in miRNA maturation, but also open up a new therapeutic strategy to treat various diseases that are linked to overexpression of G-rich miRNAs.

 描述(申请人提供)：microRNAs(MiRNAs)是一种短的寡核苷酸，调节人类一半以上的蛋白质编码基因的表达。MicroRNAs与常见的人类疾病有关，如癌症、免疫紊乱、病毒感染和神经退化。由于miRNA水平的升高往往与各种疾病状态有关，因此miRNA的生物发生是一个严格调控的过程。DICER介导的前体miRNA(Pre-miRNA)在细胞质中的成熟是miRNA生物发生中的关键步骤，RNase Disher识别并内部切割Pre-miRNA茎环以产生成熟的miRNA。目前的教条是，所有的前miRNAs都采用茎环结构，这是Dester精确成熟所必需的。因此，除了规范的茎环结构外，还没有积极考虑其他替代的二级结构用于前miRNAs。然而，在对所有已知的人前miRNA和成熟miRNA序列进行综合分析后，我们分别鉴定了298和179个序列，它们有形成稳定的G-四链(GQ)的倾向，作为替代的RNA二级结构。在这个建议中，我们打算详细研究Gq结构在miRNA生物发生中的作用，以及小分子通过靶向Gq结构控制miRNA前成熟的能力。利用在多种疾病中过度表达的临床重要miRNA(Pre-miRNA92b)，我们显示了在生理相关K+浓度下Gq的结构共存和Pre-miRNA的典型茎环结构。此外，我们的初步数据表明，GQ的形成抑制了Dird介导的人前miRNA92b的成熟。鉴于这些发现，我们假设GQ结构在miRNA成熟前和成熟的双链miRNA链分离中都在miRNA的生物发生中发挥关键作用。我们还假设，与前miRNA中的GQS结合的小分子可以调节其成熟。这些假说将在以下特定目标下进行研究：(I)确定Gq对使用人Pre-miRNA92b的DICER介导的Pre-miRNA成熟的影响；(Ii)描述成熟miRNAs中Gq的形成是否导致miRNA双链的结构解离；以及(Iii)确定针对Pre-miRNA中非正则结构的小分子是否能调节其在体外和人类细胞中的成熟。这些特定目标的完成将揭示替代二级结构在miRNA生物发生中的重要性，这反过来将导致更好地理解miRNA水平如何控制各种疾病。小分子靶向GQs将是控制内源性miRNA生物发生的新途径，可用于开发新的治疗方法。这一全面的研究不仅将为我们目前对RNA结构在miRNA成熟中的作用的理解增加一个新的维度，而且还将开辟一种新的治疗策略来治疗与富含G的miRNAs过度表达有关的各种疾病。