Molecular mechanisms for regulating microRNA levels in metazoans

项目总结 基因表达是从dna到rna再到蛋白质的遗传信息流，它涉及到一种微妙的调控机制。 一组称为microRNAs(MiRNAs)的非编码小RNA。失调的miRNA网络一直是 在各种疾病状态下观察，包括癌症、发育缺陷和神经退行性变 疾病。在这些疾病状态下，miRNA的丰度可能会在降解或 生物发生的步骤。靶向miRNA降解(TDMD)是特定miRNAs 当与它们的目标RNA广泛碱基配对时，会发生降解。TDMD如何有助于 由于缺乏可诱导miRNA的已知靶向RNA(或触发物)，不同的疾病尚不清楚 退化。另一方面，Dier在将前体miRNAs加工成 成熟的miRNA。Dester缺陷促进肿瘤发生，但不同的miRNAs如何受Low影响 癌症中的迪特尔水平仍然是个问题。为了解决这些知识差距，我们将探索分子 后生动物中特定miRNA丰度的调节机制侧重于三个项目： 1.扩展后生动物TDMD触发器的曲目。 我们已经建立了CLASH，一种生化和生物信息学相结合的管道，用于TDMD的鉴定 在培养的细胞中触发。我们将进一步完善CLASH应用于模式生物识别 保守和必要的TDMD触发器。 2.研究了有效TDMD触发器的性质。 已知的TDMD触发序列集中在mRNA 3‘非编码区和非编码区 RNA。我们将研究触发转录本的序列、结构和非编码的重要性 诱导TDMD的特性。 3.细胞miRNAs的差异处理。 在工程化的结直肠癌HCT116细胞系中，可以通过以下方式微调Dier的表达 多西环素后，我们将确定不同表达水平的miRNA图谱。的决定因素 单个miRNAs对DICER的差异反应将通过体外DICER切割试验和 MiRNA结合蛋白的鉴定。 我们独特的实验方法将为开发新的治疗方法提供基础 TDMD途径的调控和Disher作用。在这个项目中获得的技术和经验将 让我们的团队为未来探索调控小RNA必不可少的分子机制的研究做好准备 在正常组织和疾病组织中的基因表达。

PROJECT SUMMARY Gene expression, the genetic information flow from DNA to RNA to protein, involves delicate regulation by a group of small non-coding RNAs called microRNAs (miRNAs). Dysregulated miRNA networks have been observed in a variety of disease states including cancer, developmental defects, and neurodegenerative diseases. In these disease states, the miRNA abundance can be affected at either the degradation or the biogenesis steps. Target-directed miRNA degradation (TDMD) is a mechanism by which specific miRNAs undergo degradation when extensively base-paired with their target RNAs. How TDMD may contribute to different diseases is unclear due to the paucity of known target RNAs (or triggers) that can induce miRNA degradation. On the other hand, Dicer functions as the central enzyme in processing precursor miRNAs into mature miRNAs. Dicer deficiency promotes tumorigenesis, but how different miRNAs are affected by lower levels of Dicer in cancer remains a question. To address these knowledge gaps, we will probe the molecular regulatory mechanisms of specific miRNA abundance in metazoans focusing on three projects: 1. Expand the repertoire of metazoan TDMD triggers. We have established CLASH, a combined biochemical and bioinformatics pipeline, for identification of TDMD triggers in cultured cells. We will further improve CLASH for application in model organisms to identify conserved and essential TDMD triggers. 2. Investigate the properties of effective TDMD triggers. Known TDMD trigger sequences are concentrated in conserved regions of the mRNA 3′ UTR and non-coding RNAs. We will investigate the importance of trigger transcripts sequences, structure and the non-coding properties in inducing TDMD. 3. Differential Dicer processing of cellular miRNAs. In an engineered colorectal cancer HCT116 cell line, in which Dicer expression can be fine-tuned by doxycycline, we will determine the miRNA profile at different Dicer expression levels. Determinants for differential responsiveness to Dicer of individual miRNAs will be probed by in vitro Dicer cleavage assays and identification of miRNA binding proteins. Our unique experimental approaches will provide the basis for developing novel therapeutics through modulation of the TDMD pathway and Dicer action. Techniques and experience acquired in this project will prepare our group for future research in exploring molecular machineries that regulate small RNAs essential for gene expression in normal and diseased tissue.