Targeted regulation of transcript stability through RNA methylation and intron retention

项目总结 细胞依赖于蛋白质在空间和时间上的精确表达来执行关键的生物学过程。 因此，破译调控基因组是开发准确的细胞模型的关键一步。 行为。虽然转录网络已经被广泛研究，但转录后调控程序 在很大程度上仍然没有特征。最近，我们发现了双链RNA结合蛋白TARBP2 作为RNA稳定性的全球调节器(Goodarzi等人《自然》，2014)。然而，潜在的分子 这种非典型的TARBP2途径调节RNA衰退的机制尚不清楚。基座 根据我们的观察，TARBP2广泛结合到其目标转录本的内含子区域，我们 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，TARBP2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，有RNA甲基转移酶复合体和 核监视机器。我们的初步发现有力地支持了TARBP2结合模型 导致RNA甲基转移酶复合体的招募和随后的TARBP2-甲基化 结合内含子。然后，仍未剪接的甲基化内含子被靶向RNA外显体，用于 通过TARBP2和核蛋白TPR之间的相互作用而降解。 为了评估这个模型的准确性，我们将进行核RNA测序来测量内含子在 这条途径的不同组成部分的存在和不存在。首先，我们将评估TARBP2的作用 RNA甲基化中的结合及其对剪接的影响。然后，我们将寻找核燃料的成分 与TARBP2结合的内含子降解转录本的监视机制。我们还将执行上位法 为这一过程建立通路结构的实验。在每一步，除了完整的- 转录测量，我们将使用报告构建和CRISPR介导的基因组编辑来测试 TARBP2结合、RNA甲基化和剪接在RNA衰变中的必要性和充分性。 这项研究的成功完成将导致一种新的调控途径的特征 使用靶向RNA甲基化和协调内含子保留来调节RNA丰度。这项研究 这里提出的建议建立在我们多年来对基因转录后调控的多学科研究基础上 表达(Goodarzi等人，Cell 2015,2016；Goodarzi等人，自然，2012,2014)。我们的初步结果， 大力支持我们建议的模型，为 这项提议。鉴于我们在计算和实验生物学方面的深厚背景，以及 由加州大学旧金山分校的合作者提供的支持，我们处于解决这一项目的理想位置。

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.