Targeted regulation of transcript stability through RNA methylation and intron retention

项目摘要 细胞依赖于蛋白质在空间和时间上的精确表达来进行关键的生物过程。 因此，解码调节基因组是开发准确的细胞模型的关键一步 行为虽然转录网络已被广泛研究，转录后调控程序， 基本上没有特征。最近，我们发现了双链RNA结合蛋白TARBP 2 作为RNA稳定性的全局调节剂（Goodarzi等人，Nature，2014）。然而， 这种非经典的TARBP 2途径调节RNA衰变的机制是未知的。基于 根据我们观察到的TARBP 2与其靶转录物的内含子区域广泛结合的结果， 假设并成功地证明了这种衰变途径位于原子核中。我们也 结果表明，TARBP 2与细胞核和细胞质中的不同蛋白质组相互作用。有趣的是， 在核特异性相互作用中，RNA甲基转移酶复合物和 核监视机器。我们的初步研究结果强烈支持一个模型，其中TARBP 2结合 导致RNA甲基转移酶复合物的募集和随后的TARBP 2-甲基化。 结合内含子保持未剪接的甲基化内含子然后靶向RNA外泌体， 通过TARBP 2和核蛋白TPR之间的相互作用降解。 为了评估该模型的准确性，我们将进行核RNA测序，以测量内含子保留， 该途径不同组分的存在和不存在。首先，我们将评估TARBP 2的作用 RNA甲基化的结合及其对剪接的影响。然后，我们将寻找核武器的组成部分， 监控机制，降解转录与TARBP 2结合的内含子。我们还将执行上位 实验来建立这个过程的路径结构。在每一步，除了整个- 转录组学测量，我们将使用报告构建体和CRISPR介导的基因组编辑来测试 RNA降解中TARBP 2结合、RNA甲基化和剪接的需要和充分性。 这项研究的成功完成将导致一种新的调控途径的表征， 使用靶向RNA甲基化和协调的内含子保留来调节RNA丰度。研究 基于我们多年来对基因转录后调控的多学科研究， 表达（Goodarzi等，Cell 2015，2016; Goodarzi等，Nature 2012，2014）。我们的初步结果， 强烈支持我们提出的模式，为跨学科方法提供了坚实的基础， 这个提议。鉴于我们在计算和实验生物学方面的强大背景，以及我们的专业知识和 在UCSF的合作者的支持下，我们非常适合解决这个项目。

PROJECT SUMMARY Cells rely on spatially and temporally precise expression of proteins to carry out key biological processes. Thus, decoding the regulatory genome is a crucial step towards developing accurate models of cellular behavior. While transcriptional networks have been widely studied, post-transcriptional regulatory programs remain largely uncharacterized. Recently, we discovered the double-stranded RNA-binding protein TARBP2 acts as a global regulator of RNA stability (Goodarzi et al. Nature, 2014). However, the underlying molecular mechanisms through which this non-canonical TARBP2 pathway regulates RNA decay were unknown. Based on our observation that TARBP2 binds extensively to the intronic regions of its target transcripts, we hypothesized and successfully demonstrated that this decay pathway is located in the nucleus. We also showed that TARBP2 interacts with different sets of proteins in the nucleus and the cytoplasm. Interestingly, among the nucleus-specific interactions were key components of the RNA methyltransferase complex and the nuclear surveillance machinery. Our preliminary findings strongly support a model in which TARBP2 binding results in the recruitment of the RNA methyltransferase complex and the subsequent methylation of TARBP2- bound introns. Methylated introns, which remain unspliced, are then targeted to the RNA exosome for degradation through an interaction between TARBP2 and the nucleoprotein TPR. To assess the veracity of this model, we will perform nuclear RNA sequencing to measure intron retention in the presence and absence of different components of this pathway. First, we will assess the role of TARBP2 binding in RNA methylation and its impact on splicing. Then, we will search for the components of the nuclear surveillance machinery that degrade transcripts with TARBP2-bound introns. We will also perform epistasis experiments to establish the pathway structure for this process. At every step, in addition to whole- transcriptomic measurements, we will use reporter constructs and CRISPR-mediated genome editing to test the requirement and sufficiency of TARBP2 binding, RNA methylation, and splicing in RNA decay. The successful completion of this study will result in the characterization of a novel regulatory pathway that uses targeted RNA methylation and orchestrated intron retention to modulate RNA abundance. The study proposed here builds on our years of multidisciplinary research on post-transcriptional regulation of gene expression (Goodarzi et al, Cell 2015, 2016; Goodarzi et al, Nature 2012, 2014). Our preliminary results, which strongly support our proposed model, provide a strong foundation for the interdisciplinary approach outlined in this proposal. Given our strong background in computational and experimental biology, and the expertise and support provided by our collaborators at UCSF, we are ideally situated to tackle this project.