SGER: Development of Methods for Analysis of Small Non-coding Transcrips by MPSS

该项目的目标是开发和测试方法，以比以前更大的规模分离，克隆和测序小RNA分子（21至24个核苷酸）。这种大小的RNA在许多真核生物中具有特定的生物活性。 初步数据表明，这些微小的RNA分子中的大多数是小干扰RNA（siRNA）或microRNA（miRNA）。 这些小RNA可以通过抑制它们可以碱基配对的较长RNA（如mRNA）的合成或功能来关闭基因。它们通过将蛋白质复合物引导到RNA或编码RNA的DNA上来实现这一点。以这种方式，siRNA可以抑制mRNA的合成或导致mRNA的降解，而miRNA可以抑制mRNA翻译成蛋白质的能力。 与siRNA类似，miRNA也可以引起mRNA的加速降解。 siRNA和miRNA分子的鉴定和/或预测受到了表征这些分子的有限实验数据的阻碍。 高通量方法在该项目中的应用将促进这些分子的鉴定，并提供特定组织中特定小RNA分子丰度的定量估计。 小RNA序列与基因组DNA的比较可以鉴定这些RNA片段的来源和/或靶标。 该方法将广泛适用于许多真核生物，并可用于识别和测量响应于发育，生物或非生物线索而调节的小RNA。 在本项目中，将开发和实施通过MPSS对这些小RNA分子进行分离、克隆和测序的方法，并将启动数据表征和分析的生物信息学方法。 这些方法将在多种处理条件下在两种植物物种中进行测试。 对于数据的基因组分析，将使用现有的定制数据库和用于MPSS数据可视化的网络界面（http：//mpss.udel.edu/at）。 这些数据将通过这个网络界面公开。

The goal of this project is to develop and test methods to isolate, clone and sequence small RNA molecules (21 to 24 nucleotides) on a larger scale than previously possible. RNAs of this size have specific biological activities in many eukaryotes. Preliminary data suggest that most of these tiny RNA molecules are small interfering RNAs (siRNAs) or microRNAs (miRNAs). These small RNAs can turn off genes by inhibiting the synthesis or function of longer RNAs, like mRNAs, that they can base pair with. They do this by guiding protein complexes to the RNA or the DNA that encodes it. In this manner, siRNAs can inhibit the synthesis or cause degradation of mRNAs, whereas miRNAs can inhibit the ability of mRNAs to be translated into protein. miRNAs can also cause accelerated degradation of the mRNAs, similar to siRNAs. The identification and/or prediction of siRNA and miRNA molecules have been hampered by limited experimental data that characterize these molecules in depth. The application of high-throughput approaches in this project will facilitate the identification of these molecules and provide quantitative estimates of abundance for specific small RNA molecules in a given tissue. Comparisons of the small RNA sequences to genomic DNA may identify the source and/or target of these RNA fragments. The methodology will be broadly applicable in many eukaryotes and can be used to identify and measure small RNAs that are regulated in response to developmental, biotic or abiotic cues. In this project, methods for the isolation, cloning and sequencing by MPSS of these small RNA molecules will be developed and implemented, and bioinformatics methods for the characterization and analysis of the data will be initiated. These methods will be tested in two plant species under multiple treatment conditions. For the genomic analysis of the data, an existing customized database and web interface for the visualization of MPSS data will be used (http://mpss.udel.edu/at). The data will be made public via this web interface.