Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.
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.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
Brian J
BJ
Belmont
Jacquin C
JC
Niles
D020413:3' Untranslated Regions / Q000187:drug effects; D020121:5' Untranslated Regions / Q000187:drug effects; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D042541:Intracellular Space / Q000187:drug effects* / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D052157:Aptamers, Nucleotide; D012333:RNA, Messenger; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; D013752:Tetracycline