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

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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.