Regulatory roles for the Integrator complex and circular RNAs

Integrator 复合物和环状 RNA 的调节作用

• 批准号: 10553413
• 负责人: Jeremy E Wilusz
• 金额: $ 20.48万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10553413
• 关键词: Regulatory; roles; Integrator; complex; circular

PROJECT SUMMARY/ABSTRACT For a protein-coding gene to perform its cellular function, it must first generate RNA transcripts that are expressed at the appropriate level and properly processed. This is no small feat when one considers that RNA polymerase II can prematurely terminate and that nascent transcripts can be acted upon by a variety of RNA processing machines, including ones that yield mature transcripts lacking a canonical 5' cap or 3' poly(A) tail. A major focus of our laboratory has thus been to identify and characterize novel “non-canonical” processing pathways that can act on nascent RNAs. Here, we propose to build upon our recent work to study two such mechanisms that are widely employed across eukaryotic genomes. First, we will mechanistically dissect how the Integrator (Int) complex catalyzes premature transcription termination at hundreds of protein-coding genes. Integrator was long known to be critical for the biogenesis of small nuclear RNAs (snRNAs), but we recently showed that Integrator also binds to many protein-coding loci and attenuates production of their full- length mRNAs, in some cases by more than 100-fold. This is because the IntS11 RNA endonuclease directly cleaves nascent mRNAs, triggering degradation of the transcripts and premature transcription termination. Nevertheless, it remains poorly understood how Integrator is assembled, regulated, and recruited to protein- coding genes as most of the other subunits in the complex have no known function and lack obvious paralogs or known protein domains. Our preliminary data indicate that non-catalytic Integrator subunits have distinct roles at snRNA vs. protein-coding gene loci, and thus we will characterize in detail how these subunits are recruited and function. Crosslinking mass spectrometry will further be used to define physical interfaces between Integrator subunits, thereby revealing novel insights into how Integrator is globally assembled and controlled. Second, we will investigate why many protein-coding genes generate circular RNAs with covalently linked ends. Some of these non-canonical transcripts are greater than 10-fold more abundant than their associated linear mRNAs. This suggests the main function of these genes may be to produce circular RNAs, but the physiological functions of almost all mature circular RNAs remain unknown. We thus will use high-throughput screening coupled to detailed biochemical studies to identify critical functions for circular RNAs and their underlying molecular mechanisms. We further will systematically identify factors that modulate circular RNA levels, especially post-transcriptionally, as very little is currently known about how the fate and decay rates of these transcripts are controlled. Characterization of these key regulatory mechanisms will not only provide important insights into endogenous circular RNAs, but may also ultimately enable circular RNAs to become novel long-lasting therapeutic modalities. In total, these innovative studies will reveal new, fundamental insights into how the Integrator complex and circular RNAs are regulated and control protein- coding gene outputs to impact normal and diseased states.

项目总结/摘要 蛋白质编码基因要发挥其细胞功能，必须首先产生RNA转录本， 在适当的水平上表达并适当处理。这是一个不小的壮举，当一个人认为，RNA 聚合酶II可以过早终止，新生的转录物可以被各种RNA作用 处理机器，包括产生缺乏典型5'帽或3'聚腺苷酸尾的成熟转录物的机器。一 因此，我们实验室的主要重点是识别和表征新的“非规范”处理 可以作用于新生RNA的途径。在这里，我们建议建立在我们最近的工作，研究两个这样的 这些机制在真核生物基因组中广泛使用。首先，我们将机械地剖析 整合子（Int）复合物催化数百个蛋白质编码区的过早转录终止， 基因.整合子对于小核RNA（snRNA）的生物发生很重要，但我们 最近表明，整合剂也结合到许多蛋白质编码基因座，并减弱其完整的生产， 长度的mRNA，在某些情况下超过100倍。这是因为IntS 11 RNA内切酶直接 切割新生的mRNA，引发转录物的降解和过早的转录终止。 然而，人们对Integrator是如何组装、调节和招募到蛋白质中的仍然知之甚少。 编码基因和复合体中的大多数其他亚基一样，功能未知，缺乏明显的旁系同源物 或已知的蛋白质结构域。我们的初步数据表明，非催化整合子亚基具有不同的 在snRNA与蛋白质编码基因位点的作用，因此，我们将详细描述这些亚基是如何被 招募和运作。交联质谱法将进一步用于定义物理界面 在Integrator子单元之间，从而揭示了Integrator如何全局组装的新见解， 控制。其次，我们将研究为什么许多蛋白质编码基因产生环状RNA， 共价连接的末端。这些非经典转录物中的一些比非经典转录物的丰度高10倍以上。 与之相关的线性mRNA这表明这些基因的主要功能可能是产生循环， 然而，几乎所有成熟的环状RNA的生理功能仍然未知。因此，我们将使用 高通量筛选结合详细的生化研究，以确定环状RNA的关键功能 及其潜在的分子机制。我们将进一步系统地识别调节 环状RNA水平，特别是转录后水平，因为目前很少有人知道如何命运和 这些转录物的衰变速率是受控的。这些关键调控机制的特征将不会 这不仅为内源性环状RNA提供了重要的见解，而且还可能最终使环状RNA能够 成为一种新型的长效治疗方式。总之，这些创新的研究将揭示新的， 对整合子复合物和环状RNA如何调控和控制蛋白质的基本见解- 编码基因输出影响正常和疾病状态。