A systematic analysis of mir-34 function in C. elegans

描述(申请人提供)：动物miRNAs在调节许多发育过程中发挥作用(Krousterman&Plasterk，2006)，并与许多人类疾病有关(Garber，2006)。尽管它们在发育和疾病中发挥着重要作用，但对于大多数microRNAs来说，很少有靶点得到实验验证。本研究将集中于一个高度保守的microRNA，它已被证明在脊椎动物的肿瘤发生和肿瘤抑制中发挥作用，mir-34(Chang等人，2007年；他等人，2007年)，并使用几种补充技术来确定该microRNA在线虫中的作用。这项研究将通过计算、生化和分子技术确定mir-34的靶标。在与han和Ding实验室的合作下，我开发了一种计算靶标预测方法，该方法根据生物化学纯化的microRNA靶标中丰富的特征对microRNA结合位点进行评分(Hammell等人，提交于2008年)。这项研究将以计算研究的结果为基础，专注于一种名为mir-34的microRNA，并通过确定功能靶标和错误调节这些靶标基因的表型后果来探索其在线虫中的功能作用。由于mir-34在线虫中没有家族成员，仍然没有明显的表型，我还将通过计算识别和遗传验证协作调节mir-34靶基因的microRNAs。初步证据表明，mir-34突变体在致敏背景下确实有明显的表型，其中一个线虫Argonaute基因也被缺失，这一合作microRNAs的假说得到了支持。我将通过为我们的计算方法中预测最多的基因构建翻译记者来测试这种microRNA的前10个预测目标。我将在mir-34突变背景下，通过免疫沉淀(IP)RNA诱导沉默复合体(RISC)中的蛋白质来生化纯化mir-34的靶标。张某等人。(2007)的研究表明，RISC-IP方法和co-IP mRNA的微阵列杂交可以成功地回收线虫中数千个microRNA靶标mRNAs。在mir-34突变的蠕虫中重复RISC-IP方法应该会导致RISC相关mRNA池中mir-34靶点的丢失。最后，我将使用遗传上位性分析来确定mir-34是否足以调节其识别的靶标，或者在缺少mir-34功能的情况下，其他microRNAs是否正在合作调节一些靶标。从这项研究中收集到的任何信息也可以反馈到目标预测分析中，以改进我们的方法。

DESCRIPTION (provided by applicant): Animal miRNAs play a role in regulating many developmental processes (Kloosterman & Plasterk, 2006) and have been implicated in many human diseases (Garber, 2006). Despite their important roles in development and disease, few targets have been experimentally validated for most microRNAs. This study will focus a single highly conserved microRNA that has been shown to play a role in oncogenesis and tumor suppression in vertebrates, mir-34 (Chang et al., 2007; He et al., 2007), and use several complementary techniques to identify the role of this microRNA in the nematode C. elegans. This study will identify the targets of mir-34 through computational, biochemical, and molecular techniques. In collaboration with the Han and Ding labs, I have developed a computational target prediction method, which scores microRNA binding sites based on features enriched in biochemically purified microRNA targets (Hammell et al., Submitted 2008). This study will build on the results of that computational study by focusing on one microRNA, mir-34, and exploring its functional role in C. elegans through identifying functional targets and the phenotypic consequences of misregulating these target genes. Since mir-34 has no family members in C. elegans yet still has no overt phenotype, I will also computationally identify and genetically validate microRNAs that cooperate to regulate mir-34 target genes. This hypothesis of cooperating microRNAs is supported by preliminary evidence showing that a mir-34 mutant does have overt phenotypes in a sensitized background where one of the C. elegans Argonaute genes has also been deleted. I will test the top 10 predicted targets of this microRNA by constructing translational reporters for the top predicted genes from our computational method. I will biochemically purify targets of mir-34 by immuno- precipitating (IP) proteins in the RNA induced silencing complex (RISC) in a mir-34 mutant background. Zhang et al. (2007) has shown that a RISC-IP method followed by microarray hybridization of co-IP mRNA can successfully recover thousands of microRNA target mRNAs in C. elegans. Repeating the RISC-IP method in worms mutant for mir-34 should result in the loss of mir-34 targets from the pool of RISC- associated mRNA. Finally, I will use genetic epistasis analysis to identify whether mir-34 is sufficient to regulate its identified targets or whether other microRNAs are cooperating to regulate some targets in the absence of mir-34 function. Any information gleaned from this study can also feed back into the target prediction analysis to improve our methods.