Regulatory roles for the Integrator complex and circular RNAs

项目摘要/摘要 为了让蛋白质编码基因发挥其细胞功能，它必须首先产生 以适当的水平表达并得到适当的处理。当人们考虑到RNA时，这不是一个小壮举 聚合酶II可以过早终止，新生转录物可以被各种RNA作用 加工机器，包括那些产生没有典型的5‘帽或3’聚(A)尾的成熟转录本的机器。一个 因此，我们实验室的主要工作重点是识别和表征新的“非规范”工艺 可以作用于新生RNA的途径。在这里，我们建议在我们最近工作的基础上，研究两个这样的 广泛应用于真核生物基因组的机制。首先，我们将机械地剖析如何 整合子(Int)复合体催化数百个蛋白质编码的转录提前终止 基因。长期以来，整合子被认为对小核RNA(SnRNAs)的生物发生至关重要，但我们 最近发现，整合子还与许多蛋白质编码基因座结合，并抑制它们的完整编码基因的产生. 长度的mRNA，在某些情况下增加了100倍以上。这是因为IntS11 RNA内切酶直接 切割新生的mRNAs，引发转录产物的降解和提前转录终止。 然而，整合子是如何组装、调节和招募到蛋白质中的仍然知之甚少。 编码基因，因为复合体中的大多数其他亚基功能未知，缺乏明显的平行关系 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明显的 因此，我们将详细描述这些亚基是如何 招募并发挥作用。将进一步使用交联质谱来定义物理界面 ，从而揭示了集成商是如何在全球范围内组装和 控制住了。第二，我们将研究为什么许多编码蛋白质的基因会产生环状RNA 共价连接的末端。其中一些非规范转录本的丰度比 它们相关的线性mRNAs。这表明这些基因的主要功能可能是产生环状 但几乎所有成熟的环状RNA的生理功能仍不清楚。因此，我们将使用 结合详细生化研究的高通量筛选，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确定调节因素 环状RNA水平，特别是转录后水平，因为目前对命运和 这些转录产物的衰减率是受控制的。对这些关键监管机制的定性将不会 不仅提供了对内源性环状RNA的重要见解，而且最终也可能使环状RNA能够 成为新的长效治疗方式。总而言之，这些创新研究将揭示新的、 对整合子复合体和环状RNA如何调控和控制蛋白质的基本见解- 编码基因输出以影响正常和疾病状态。

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.