In vivo evolution of an RNA-based transcriptional silencing domain in S. cerevisiae.
Starting from a random RNA library expressed in yeast cells, we evolved an RNA-based transcriptional silencing domain with potency comparable to that observed when Sir1, a known silencing protein, is localized to a promoter. Using secondary-structure predictions and site-directed mutagenesis, we dissected the functional domains of the most active evolved RNA transcriptional silencer. Observed RNA-based silencing was general, rather than gene specific, and the origin recognition complex was required for full activity of the evolved RNA. Using genetic studies, we demonstrated that the RNA-based silencer acts through a Sir protein-dependent mechanism. Our results highlight the value of evolving RNA libraries as probes of biological processes and suggest the possible existence of natural RNA-based, RNAi-independent gene silencers.
Kehayova
Polina D
PD
Howard Hughes Medical Institute and Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 01238, USA.
Liu
David R
DR
D019020:Directed Molecular Evolution; D020868:Gene Silencing; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D012313:RNA