Abstract
Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis–elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs), which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically–inducible RNA–protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i) the Tet Repressor protein (TetR) genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii) a target transcript containing genetically encoded TetR–binding RNA aptamers. TetR–fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.
Highlights
Specific subcellular RNA localization has long been recognized as a central mechanism regulating important biological processes, such as mating type switching in Saccharomyces cerevisiae, defining body axis polarity in Drosophila and C. elegans, fibroblast and neuronal growth cone migration, synaptic plasticity, and storage of maternally–derived transcripts [for some pertinent reviews, see: [1,2,3]]
Through protein–protein interactions scaffolded by the RNA-binding proteins (RBPs), these ribonucleoprotein (RNP) complexes can be actively transported to or become entrapped within specific subcellular regions [3]
Saccharomyces cerevisiae was used as a model context for this work, as many of the molecular details underlying its natural RNA localization machinery have been elucidated
Summary
Specific subcellular RNA localization has long been recognized as a central mechanism regulating important biological processes, such as mating type switching in Saccharomyces cerevisiae, defining body axis polarity in Drosophila and C. elegans, fibroblast and neuronal growth cone migration, synaptic plasticity, and storage of maternally–derived transcripts [for some pertinent reviews, see: [1,2,3]]. As we gain a better understanding of how RNA localization shapes cellular function, opportunities to use this knowledge in design–oriented applications in areas such as synthetic biology and neurobiology will emerge Towards this long–term objective, previous efforts have used endogenously recognized cis–elements introduced into target transcripts that are. Heterologous cis–elements based on MS2 and boxB RNA binding sites have been encoded into target transcripts and co–expressed with protein fusions between the MS2 coat or lN proteins and endogenous localization effector proteins [19,20] This approach can permit regulation of transcript localization by using inducible promoters to control synthesis of the RBP–effector protein fusion [21]. Due to the inherent flexibility of this system, we envision that it can serve as a platform for both recapitulating and creating more complex and functionally relevant RNA localization schemes in a variety of organisms
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