Higher-order RNA Structural Requirements and Small Molecule Induction of Tombusvirus Subgenomic mRNA Transcription

Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3

^* To whom correspondence should be addressed. Email: kawhite{at}yorku.ca.

	Abstract

Subgenomic (sg) mRNAs are small viral messages that are synthesized by polycistronic positive-strand RNA viruses to allow for the translation of certain viral proteins. Tombusviruses synthesize two such sg mRNAs via a premature termination mechanism. This transcriptional process involves the viral RNA-dependent RNA polymerase terminating minus-strand RNA synthesis prematurely at internal RNA signals during copying of the viral genome. The 3'-truncated minus-strand RNAs generated by the termination events then serve as templates for sg mRNA transcription. A higher-order RNA structure in the viral genome, located just upstream from the termination site, is a critical component of the RNA-based polymerase attenuation signal. Here, we have analyzed the role of this RNA structure in mediating efficient sg mRNA2 transcription. Our results: (i) define the minimum overall thermodynamic stability required for an operational higher-order RNA attenuation structure; (ii) show that the distribution of stability within an attenuation structure affects its function; (iii) establish that an RNA quadruplex structure can act as an effective attenuation structure; (iv) prove that the higher-order RNA structure forms and functions in the plus strand; (v) provide evidence that a specific attenuation structure-binding protein factor is not required for transcription; and (vi) demonstrate that sg mRNA transcription can be controlled artificially through small molecule activation using RNA aptamer technology. These findings provide important new insights into the premature termination mechanism and present a novel approach to regulate the transcriptional process.