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

描述（由申请人提供）：动物miRNA在调节许多发育过程中起作用（Kloosterman和Plasterk，2006），并且与许多人类疾病有关（Garber，2006）。尽管它们在发育和疾病中起着重要作用，但大多数microRNA的靶点很少得到实验验证。本研究将集中于已显示在脊椎动物中的肿瘤发生和肿瘤抑制中起作用的单一高度保守的微小RNA，mir-34（Chang等人，2007; He等人，2007），并使用几种互补技术来鉴定这种microRNA在线虫C.优雅这项研究将通过计算，生物化学和分子技术来确定mir-34的靶点。与Han和Ding实验室合作，我开发了一种计算靶点预测方法，该方法基于在生物化学纯化的microRNA靶点中富集的特征对microRNA结合位点进行评分（Hammell et al.，2008年提交）。这项研究将建立在计算研究的结果基础上，重点关注一种microRNA，mir-34，并探索其在C.通过识别功能靶标和这些靶基因失调的表型后果来研究线虫。因为米尔-34在C区没有家人。虽然线虫仍然没有明显的表型，但我也将通过计算识别和遗传验证协同调节mir-34靶基因的microRNA。初步证据支持了这种合作microRNA的假设，表明mir-34突变体在致敏背景中确实具有明显的表型，其中一种C。线虫Argonaute基因也被删除了。我将通过我们的计算方法为预测最高的基因构建翻译报告基因来测试这种microRNA的前10个预测靶点。我将通过在mir-34突变体背景中的RNA诱导沉默复合物（RISC）中的免疫沉淀（IP）蛋白来生物化学纯化mir-34的靶标。Zhang等人（2007）已经表明，RISC-IP方法随后是co-IP mRNA的微阵列杂交，可以成功地在C.优雅在mir-34的蠕虫突变体中重复RISC-IP方法应导致mir-34靶标从RISC相关mRNA库中丢失。最后，我将使用遗传上位性分析来确定mir-34是否足以调节其确定的靶标，或者其他microRNA是否在mir-34功能缺失的情况下合作调节某些靶标。从这项研究中收集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.