A Non-Canonical RNA Structure Regulates Pre-miRNA Maturation

 描述（申请人提供）：微小RNA（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。MicroRNA与常见的人类疾病如癌症、免疫紊乱、病毒感染和神经变性有关。由于miRNA水平升高通常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，其中RNase酶Dicer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pre-miRNA都采用茎环结构，这是Dicer精确成熟所需的。因此，除了典型的茎环结构，没有其他替代的二级结构被积极考虑用于前体miRNA。然而，在对所有已知的人类pre-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，它们具有形成稳定的G-四链体（GQ）作为替代RNA二级结构的倾向。在这个提议中，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结构来控制pre-miRNA成熟的能力。使用在各种疾病中过表达的临床上重要的miRNA（pre-miRNA 92 b），我们显示了在生理相关的K+浓度下GQ和pre-miRNA的典型茎环结构的结构共存。此外，我们的初步数据显示GQ形成抑制Dicer介导的人pre-miRNA 92 b的成熟。鉴于这些发现，我们假设GQ结构在前miRNA成熟和成熟双链体miRNA链分离的miRNA生物发生中起着至关重要的作用。我们还假设小分子与pre-miRNA中的GQ结合可以调节其成熟。这些假设将在以下特定目标下进行研究：（I）使用人pre-miRNA 92 b确定GQ对Dicer介导的pre-miRNA成熟的影响，（II）描述成熟miRNA中的GQ形成是否引起miRNA双链体的结构解旋;以及（III）确定靶向pre-miRNA中非典型结构的小分子是否可以在体外和在人细胞中通过Dicer调节其成熟。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，这反过来又将导致更好地了解miRNA水平如何控制各种疾病。小分子靶向GQs将成为调控内源性miRNA生物合成的新途径，可用于开发新的治疗药物。这项全面的研究不仅将为我们目前对miRNA成熟中RNA结构作用的理解增加一个新的维度，而且还开辟了一种新的治疗策略来治疗与富含G的miRNA过表达相关的各种疾病。

 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.