Project 1 - Nuclear Gene Expression

该计划的长期目标是了解调节HIV-1核的分子机制 基因表达，并利用这一知识作为跳板来确定新的治疗靶点 干预。HIV-1生物学中的一个主要障碍是许多RNA结构和蛋白质-RNA相互作用 在细胞核中调节基因表达的机制尚不清楚。这些过程似乎是组合在一起的 自然，因此系统水平的方法必须与功能生物化学相结合，以阐明 决定HIV-1基因表达模式的分子事件。我们队(Al-Hashimi，Bieniasz，Cullen， Hargrove、Kutluay、Rouskin、Swanstrom和Tolbert)将使用病毒学方面的跨学科专业知识， 化学生物学和结构生物物理学，以确定剪接和转录控制的机制。我们 将建立在CRNA 1.0的成功基础上，包括：1)首次绘制特定结合位点的图谱 在HIV-1基因组上剪接调控蛋白；2)用一种新的方法定量测定HIV-1选择性剪接 下一代测序平台；3)发现控制HIV-1剪接的新型顺式RNA元件，并 4)测定新的HIV-1RNA剪接元件的3D结构，包括自由的和与调节分子结合的 蛋白。在这些新发现的激励下，这个项目将克服阻碍 追求对HIV-1剪接调控的理解。在CRNA 2.0中，我们将A)揭示调节蛋白质如何 因子和RNA元件串扰以建立HIV-1剪接模式；B)阐明RNA的3D结构 和调节HIV-1剪接的蛋白质-RNA复合体；c)决定RNA 结构影响剪接；以及D)开发以小分子抑制剂为靶点的HIV-1RNA的新策略。

The long-term goal of this Program is to understand the molecular mechanisms that regulate HIV-1 nuclear gene expression and to use this knowledge as a springboard to identify novel targets for therapeutic intervention. A major barrier in HIV-1 biology is that many of the RNA structures and protein-RNA interactions that regulate gene expression in the nucleus are unknown. These processes appear to be combinatorial in nature, and thus systems-level approaches must be combined with functional biochemistry in order to elucidate molecular events that determine patterns of HIV-1 gene expression. Our team (Al-Hashimi, Bieniasz, Cullen, Hargrove, Kutluay, Rouskin, Swanstrom and Tolbert) will use cross-disciplinary expertise in virology, chemical biology and structural biophysics to determine mechanisms of splicing and transcriptional control. We will build on the successes of CRNA 1.0 that include: 1) mapping for the first time binding sites of specific splicing regulatory proteins on the HIV-1 genome; 2) quantitation of the HIV-1 alternative splicing using a novel next generation sequencing platform; 3) identification of novel cis RNA elements that control HIV-1 splicing and 4) determination of the 3D structures of novel HIV-1 RNA splicing elements both free and bound to a regulatory protein. Motivated by these new discoveries, this project will overcome critical barriers that have stymied pursuit of the understanding of HIV-1 splicing regulation. In CRNA 2.0, we will A) reveal how regulatory protein factors and RNA elements cross talk to establish HIV-1 splicing patterns; B) elucidate 3D structures of RNA and protein-RNA complexes that regulate HIV-1 splicing; C) determine the mechanisms by which RNA structure affects splicing; and D) develop novel strategies to target HIV-1 RNA with small molecule inhibitors.