Structural studies with regulatory RNAs

总的来说，我们的Meg3研究回答了关于这个lncRNA的结构和活性的基本问题，为这个家族的其他lRNA的功能和机制研究提供了一个模板。这些发现也为有效利用RNA的肿瘤抑制特性用于潜在的抗癌治疗奠定了框架。Meg3RNA是到目前为止特征最好的肿瘤抑制因子lncRNA。在各种肿瘤细胞系和临床标本中，Meg3要么不表达，要么低水平表达，外源Meg3 RNA在这些细胞中的表达可以减缓细胞生长并诱导细胞凋亡。因此，在受影响的细胞中靶向恢复或过度表达Meg3 RNA，为癌症治疗提供了一条有希望的途径。我们对KSHV PAN lncRNA的结构探测数据为在各种生物背景下全面重建蛋白质-lncRNA相互作用网络铺平了道路。PAN RNA是一种高度多功能的病毒转录本，通过局部结构的RNA片段以及与几种病毒和宿主蛋白的相互作用来调节其作用。对PAN和其他KSHV lncRNAs的进一步研究将进一步阐明这种重要病原体的复杂分子生物学。对乙肝病毒(乙肝病毒)的核磁共振研究揭示了包装元件epsilon与DNA合成的启动有关的新的机制见解，为治疗干预提供了额外的途径。我们与NIAID综合研究机构合作，确定了埃博拉病毒微基因组的结构。在这里，我们证明了EBOV RNA基因组的5个启动子NCR形成了复杂的第二和第三结构域，这些结构域与宿主蛋白相互作用，是病毒复制所必需的。我们的数据表明，宿主蛋白HSPA8与一个特定的RNA基序相互作用，该基序形成了位于EBOV拖车(NT 28-37)中的茎环的右侧。这种相互作用对于EBOV微基因组复制和从cDNA中回收全长感染性病毒是必不可少的。这些研究为开发针对埃博拉病毒基因组的配体奠定了基础。该项目的工作已于21财年结束。

Collectively, our Meg3 study answers basic questions regarding structure and activity of this lncRNA, providing a template for functional and mechanistic studies of other lRNAs of this family. These findings also lay a framework for efficiently harnessing the tumor suppressive property of the RNA for potential anti-cancer therapies. Meg3 RNA is by far the best-characterized tumor suppressor lncRNA. In various cancer cell lines and clinical samples, Meg3 is either not expressed or is expressed at low levels, and exogenous expression of Meg3 RNA in such cells can slow growth and induce apoptosis. Targeted restoration or overexpression of Meg3 RNA in affected cells, therefore, offers a promising avenue for cancer treatment. Our structural probing data for KSHV PAN lncRNA pave the way for a comprehensive reconstruction of the protein-lncRNA interaction network in various biological contexts. PAN RNA is a highly multifunctional viral transcript that mediates its effects through locally structured RNA segments and interactions with several viral and host proteins. Additional studies of PAN and other KSHV lncRNAs will further elucidate the complex molecular biology of this important pathogen. NMR studies with hepatitis B virus (HBV) have revealed novel mechanistic insights into the packaging element, epsilon, as it relates to initiation of DNA synthesis, providing additional avenues for therapeutic intervention. In collaboration with the NIAID Integrated Research Facility, we have determined the structure of an Ebola virus minigenome. Herein, we demonstrated that the 5 prime NCR of the EBOV RNA genome forms complex secondary and tertiary domains that interact with a host protein and are essential for virus replication. Our data indicate that host protein HSPA8 interacts with a specific RNA motif that forms the right side of a stem-loop located in the EBOV trailer (nt 28-37). This interaction is essential for EBOV minigenome replication and recovery of full-length infectious virus from cDNA. These studies lay a foundation for development of ligands that target the Ebola virus genome. Work on this project concluded in FY21.