Higher-order RNA structural requirements and small-molecule induction of tombusvirus subgenomic mRNA transcription.
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 include the following: (i) we define the minimum overall thermodynamic stability required for an operational higher-order RNA attenuation structure; (ii) we show that the distribution of stability within an attenuation structure affects its function; (iii) we establish that an RNA quadruplex structure can act as an effective attenuation structure; (iv) we prove that the higher-order RNA structure forms and functions in the plus strand; (v) we provide evidence that a specific attenuation structure-binding protein factor is not required for transcription; (vi) we 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.
Wang
Sheng
S
Department of Biology, York University, Toronto, Ontario, Canada.
Mortazavi
Leyla
L
White
K Andrew
KA
D052157:Aptamers, Nucleotide / Q000378:metabolism*; D001483:Base Sequence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D017864:Tombusvirus / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*
D052157:Aptamers, Nucleotide; D012333:RNA, Messenger; D012367:RNA, Viral