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

感觉光感受器蛋白支持对生物生理、特征、发育和行为的入射光线的重要适应。它们在光遗传学中兼具基因编码、光门驱动的作用，能够在时空上精确和可逆地控制光对细胞状态和过程的控制。虽然不同的过程都可以通过光遗传干预，但长期以来，没有光感受器以光依赖的方式与RNA相互作用。通过从多部Nakamurella中鉴定出RNA结合的光氧电压(LOV)受体PAL，我们最近缩小了这一差距。在蓝光下，PAL序列以低纳摩尔亲和力与RNA发夹特异结合，从而支持前所未有的以RNA为中心的光遗传学应用，称为光学遗传学。在目前的合作项目中，我们试图阐明PAL中光激活RNA结合的结构和机制基础，以及最近从Nakamurella derti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，而且也预示着对光依赖的变构和信号响应的RNA：蛋白质相互作用的基本见解。利用SELEX(通过指数富集法对配体的系统进化)，RNA分子将被提高序列特异性，并分别紧密结合PAL受体的暗适应和光适应状态。通过利用这些知识，我们将实施新的光学遗传学模式来调节细菌、哺乳动物细胞和果蝇的基因表达。鉴于PAL在RNA水平上起作用，光遗传应用绕过了DNA水平，这至少提供了两个主要优势。首先，PAL引发的光依赖反应预计会比转录水平上的反应出现和停止得更快。其次，光依赖的PAL：RNA相互作用有助于与基因电路的结合和整合，无论是光调控的还是其他的，以更好的通用性和控制性来调节细胞事件。

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.