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

感觉光感受器蛋白质是生物体生理、性状、发育和行为对入射光的重要适应的基础。它们在光遗传学中兼作基因编码、光门控致动器，使光对细胞状态和过程的时空精确和可逆控制成为可能。虽然多种过程都可以接受光遗传干预，但长期以来没有光感受器以依赖光的方式与RNA相互作用。通过鉴定来自Nakamurella multipartita的rna结合光氧电压（LOV）受体PAL，我们最近缩小了这一差距。暴露在蓝光下，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和光激活RNA结合的结构和机制基础以及最近从Nakamurella deserti中发现的两个同源物。这些研究不仅将为光遗传学的应用提供信息，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。利用SELEX（配体的指数富集系统进化），RNA分子将分别与PAL受体的暗适应和光适应状态进行序列特异性和紧密结合。通过利用这些知识，我们将实现细菌、哺乳动物细胞和黑腹果蝇基因表达调控的新型光生模式。鉴于PAL在RNA水平起作用，光化学应用绕过DNA水平，这至少提供了两个主要优点。首先，PAL引发的光依赖性反应预计比转录水平上的反应更快地显现和停止。其次，依赖于光的PAL:RNA相互作用使其能够与光调节或其他遗传回路结合和整合，以更好的通用性和控制力来调节细胞事件。

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.