In vivo evolution of RNA-based activators and silencers of transcription.

摘要

In nature RNA plays multiple roles (some of which are reviewed in Chapter One), in addition to serving as a transient carrier of genetic information. We used directed evolution methods to explore the potential of RNA to probe complex biological processes. A combination of in vivo selections and random mutagenesis in S. cerevisiae led to the identification of two classes of RNAs capable of regulating transcription.; Chapter Two describes the directed evolution of RNA transcriptional activation domains, starting from random RNA libraries expressed in yeast. The most potent RNA activated transcription 53-fold higher than a three-hybrid positive control using the Gal4 activation domain and only two-fold lower than the highly active VP16 activation domain. A surprisingly large fraction of random RNAs from our library are capable of activating transcription, suggesting that nucleic acids may be well suited for binding transcriptional machinery elements normally recruited by proteins.; Chapter Three describes the evolution of an RNA-based transcriptional silencing domain. The most evolved RNA silences gene expression to a level similar to that observed when Sir1, a known silencing protein, is localized to a promoter. RNA-based silencing is general, rather than gene-dependent, and the Origin Recognition Complex (ORC) is required for full activity. Using genetic studies, we demonstrated that the RNA-based silencer acts through a Sir protein-dependent mechanism.; Chapter Four describes progress towards the elucidation of the mechanism of action of m26-11, a potent evolved RNA-based transcriptional activator. Mutagenesis combined with genetic complementation identified proteins involved in the regulation of glycolytic genes as potential targets of m26-11. Functional studies of deletion mutants and immunoprecipitation experiments suggested that the DNA-binding proteins Gcr1 and Rap1 participate in a ribonucleoprotein (RNP) complex that includes m26-11 and that this complex likely is responsible for RNA-dependent transcriptional activation.; Our findings indicate that RNA is capable of accessing the structural diversity needed to mimic a protein transcriptional activation or silencing domain. 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 activators and silencers.