Engineering Structurally Interacting RNA (sxRNA)

Francis Doyle,Salvatore Spadaro,Scott A Tenenbaum,Zachary E Wurz,Sumita Bhaduri-Mcintosh,Sameer Lapsia

doi:10.1038/srep45393

Francis Doyle, Salvatore Spadaro + Show 4 more

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RNA-based three-way junctions (3WJs) are naturally occurring structures found in many functional RNA molecules including rRNA, tRNA, snRNA and ribozymes. 3WJs are typically characterized as resulting from an RNA molecule folding back on itself in cis but could also form in trans when one RNA, for instance a microRNA binds to a second structured RNA, such as a mRNA. Trans-3WJs can influence the final shape of one or both of the RNA molecules and can thus provide a means for modulating the availability of regulatory motifs including potential protein or microRNA binding sites. Regulatory 3WJs generated in trans represent a newly identified regulatory category that we call structurally interacting RNA or sxRNA for convenience. Here we show that they can be rationally designed using familiar cis-3WJ examples as a guide. We demonstrate that an sxRNA “bait” sequence can be designed to interact with a specific microRNA “trigger” sequence, creating a regulatable RNA-binding protein motif that retains its functional activity. Further, we show that when placed downstream of a coding sequence, sxRNA can be used to switch “ON” translation of that sequence in the presence of the trigger microRNA and the amount of translation corresponded with the amount of microRNA present.

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Many RBPs associate with their RNA targets by binding to motifs with structures that contain one or more stem-loops[9,10,11]
Using the recognized rules for cis-3WJ structures[1], we demonstrate the ability to rationally design a two-piece, trans histone stem-loop (HSL)-sxRNA into a reporter message, with a regulatable, HSL-sequence that requires a targeted cellular microRNA to act as a “trigger” to form a functional trans-HSL motif, which can associate with endogenous stem-loop binding protein (SLBP)
In our earlier work we described naturally occurring interactions that resulted from trans-3WJ forming between various cellular microRNAs and histone mRNA, at the location of the HSL motif[7,8]

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Many RBPs associate with their RNA targets by binding to motifs with structures that contain one or more stem-loops[9,10,11]. The regulation of metazoan histones requires a specialized post-transcriptional mechanism using the conserved HSL motif comprised of a 16-base stem-loop structure plus 3–4 bases on both its immediate 5′and 3′flanks. Studies have demonstrated that when mRNA containing a terminal HSL is ectopically introduced into cells, it will be actively translated upon endogenous SLBP binding, and can be used to efficiently express a reporter protein[14,17]. Using the recognized rules for cis-3WJ structures[1], we demonstrate the ability to rationally design a two-piece, trans HSL-sxRNA into a reporter message, with a regulatable, HSL-sequence (the bait) that requires a targeted cellular microRNA to act as a “trigger” to form a functional trans-HSL motif, which can associate with endogenous SLBP. We show that an HSL-sxRNA can be used to regulate the translation of an upstream coding sequence and that expression correlates with the amount of triggering microRNA present in the cell

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