Structure, Function, and Optoribogenetics of RNA-Binding Blue-Light Receptors

RNA 结合蓝光受体的结构、功能和光遗传学

• 批准号: 522261769
• 负责人: Professor Dr. Günter Mayer
• 金额: --
• 依托单位: Arbeitsgruppe Biochemie I
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/522261769?language=en
• 关键词: Structure; Function; Optoribogenetics; RNA; Binding

Sensory photoreceptor proteins underpin vital adaptations to incident light of organismal physiology, traits, development, and behavior. They double as genetically encoded, light-gated actuators in optogenetics to enable the spatiotemporally precise and reversible control of cellular state and processes by light. Although diverse processes are amenable to optogenetic intervention, long no photoreceptors interacting with RNA in light-dependent manner were known. By identifying the RNA-binding light-oxygen-voltage (LOV) receptor PAL from Nakamurella multipartita, we recently closed this gap. Exposed to blue light, PAL sequence-specifically binds to RNA hairpins with low nanomolar affinity and thereby supports unprecedented RNA-centered optogenetic applications, referred to as optoribogenetics. Within the present collaborative project, we seek to elucidate the structural and mechanistic bases for light-activated RNA binding in PAL and two recently identified homologs from Nakamurella deserti. Not only will these studies inform optoribogenetic application, but also they augur fundamental insight into light-dependent allostery and signal-responsive RNA:protein interactions. Using SELEX (systematic evolution of ligands by exponential enrichment), RNA molecules will be raised that sequence-specifically and tightly bind the dark-adapted and light-adapted states, respectively, of the PAL receptors. By harnessing this knowledge, we will implement novel optoribogenetic modalities for the regulation of gene expression in bacteria, mammalian cells, and Drosophila melanogaster. Given that PAL acts at the RNA level, optoribogenetic application bypasses the DNA level which affords at least two principal advantages. First, light-dependent responses elicited by PAL are expected to manifest and cease faster than those operating at the transcriptional level. Second, the light-dependent PAL:RNA interaction lends itself to the combination and integration with genetic circuits, light-regulated or otherwise, to modulate cellular events with ever better versatility and control.

感觉光感受器蛋白支持生物体生理学、特征、发育和行为对入射光的重要适应。它们在光遗传学中兼作遗传编码的光门控致动器，使光能够在时空上精确和可逆地控制细胞状态和过程。虽然不同的过程是服从光遗传学干预，长期没有光受体与RNA相互作用的光依赖性的方式是已知的。通过鉴定来自Nakamurella multipartita的RNA结合光氧电压（LOV）受体PAL，我们最近关闭了这个缺口。暴露于蓝光下，PAL序列以低纳摩尔亲和力特异性结合RNA发夹，从而支持前所未有的以RNA为中心的光遗传学应用，称为光遗传学。在目前的合作项目中，我们试图阐明PAL和两个最近发现的Nakamurella deserti同源物中光激活RNA结合的结构和机制基础。这些研究不仅将为光生应用提供信息，而且还预示着对光依赖性变构和信号响应性RNA：蛋白质相互作用的基本见解。使用SELEX（通过指数富集的配体系统进化），将产生RNA分子，其序列特异性地且紧密地结合PAL受体的暗适应和光适应状态。通过利用这些知识，我们将实施新的optoribogenetic模式，在细菌，哺乳动物细胞和果蝇的基因表达调控。考虑到PAL在RNA水平起作用，光生应用绕过了DNA水平，这提供了至少两个主要优点。首先，由PAL引起的光依赖性反应预期比在转录水平上操作的反应更快地显现和停止。第二，光依赖性PAL：RNA相互作用使其自身与光调节或其他方式的遗传电路组合和整合，以更好的多功能性和控制来调节细胞事件。