CAREER: Global Analysis of RNA-dependent RNA Polymerase Function

知识价值。真核生物如何调节mRNA水平是生物学中的一个基本问题。早期的注意力集中在基因转录水平上的调控，但最近转录后机制因其调控的重要性而得到认可。其中一种转录后机制，RNA沉默，指导各种基因转录物、重复序列、病毒和移动元件的序列特异性调控。RNA沉默是由双链RNA （dsRNA）或自互补折叠结构的产生触发的，这些结构产生小RNA (smrna)，称为微RNA或小干扰RNA （sirna）。旨在揭示RNA沉默所需因素的遗传学研究已经确定了一类称为RNA依赖RNA聚合酶（RDRs）的酶，它们使用RNA模板合成产生sirna的dsRNA分子。越来越多的证据表明，rdr依赖的RNA沉默在控制真核生物的许多方面，从基因表达到对病原体的保护。然而，对于任何生物体，rdr依赖性RNA沉默的全面功能分析仍然缺乏。为此，本项目旨在揭示6种拟南芥rdr中所有产生smrna的底物，并确定这些rna对转录组的调控作用。这项研究也将开始功能表征这些smrna对拟南芥发育和生物学的影响。由于smrna在植物中起着如此关键的调节作用，这一发现可能对未来的农作物改良具有重要意义。更广泛的影响。除了其科学影响外，该项目还将对下一代科学家的发展和培训产生广泛的影响。该项目将为本科生，包括社区大学生，甚至高中生，提供许多参与实验室研究的机会。此外，该项目产生的数据将用于开发教学模块，重点是基于高通量测序数据的计算分析。这些教学模块将被设计为适合高中水平的学习，并将免费提供，以便许多机构的教师和学生可以利用它们。

Intellectual Merit. How eukaryotic organisms regulate mRNA levels is a fundamental question in biology. Early attention was focused regulation at the level of gene transcription, but recently post-transcriptional mechanisms have gained recognition for their regulatory importance. One such post-transcriptional mechanism, RNA silencing, directs sequence-specific regulation of various gene transcripts, repetitive sequences, viruses, and mobile elements. RNA silencing is triggered by production of double-stranded RNA (dsRNA) or self-complementary fold-back structures that give rise to small RNAs (smRNAs) known as microRNAs or small-interfering RNAs (siRNAs). Genetic studies aimed at uncovering factors required for RNA silencing have identified a class of enzymes called RNA-dependent RNA polymerases (RDRs), which use an RNA template to synthesize siRNA-producing dsRNA molecules. A growing body of evidence implicates RDR-dependent RNA silencing in controlling numerous aspects of eukaryotic biology ranging from gene expression to protection from pathogens. However, a comprehensive functional analysis of RDR-dependent RNA silencing is still lacking for any organism. To this end, this project seeks to uncover all smRNA-producing substrates of the six Arabidopsis RDRs, and to determine the regulatory impact of these RNAs on the transcriptome. This research will also begin to functionally characterize the effects of these smRNAs on Arabidopsis development and biology. Because smRNAs play such a key regulatory role in the plant, the findings could have important implications for future agricultural crop improvement.Broader impacts. In addition to its scientific impact, this project will have a broad impact on the development and training of the next generation of scientists. The project will provide many opportunities for undergraduates, including community college students, and even high school students, to participate in laboratory research. Furthermore, the data generated from this project will be used to develop teaching modules focused on computational-based analysis of high-throughput sequencing data. These teaching modules will be designed to be appropriate for high school level learning and will be made freely available, so that teachers and students at many institutions can make use of them.