In vivo evolution of an RNA-based transcriptional activator.

From random RNA libraries expressed in yeast, we evolved RNA-based transcriptional activators that are comparable in potency to the strongest natural protein activation domains. The evolved RNAs activated transcription up to 53-fold higher than a three-hybrid positive control using the Gal4 activation domain and only 2-fold lower than the highly active VP16 activation domain. Using a combination of directed evolution and site-directed mutagenesis, we dissected the functional elements of the evolved transcriptional activators. A surprisingly large fraction of 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. In addition, our work demonstrates an RNA evolution-based approach to perturbing natural cellular function that may serve as a general tool for studying selectable or screenable biological processes in living cells.

[1]  M. Wickens,et al.  Identification of RNAs that bind to a specific protein using the yeast three-hybrid system. , 1999, RNA.

[2]  K. Struhl,et al.  The glutamine-rich activation domains of human Sp1 do not stimulate transcription in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[3]  M. Wickens,et al.  A three-hybrid system to detect RNA-protein interactions in vivo. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[4]  Marvin Wickens,et al.  Analyzing mRNA-protein complexes using a yeast three-hybrid system. , 2002, Methods.

[5]  K. Struhl,et al.  Yeast transcriptional regulatory mechanisms. , 1995, Annual review of genetics.

[6]  M. Ptashne,et al.  An artificial transcriptional activating region with unusual properties. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Jun Ma,et al.  GAL4-VP16 is an unusually potent transcriptional activator , 1988, Nature.

[8]  M. Wickens,et al.  Yeast three-hybrid system to detect and analyze RNA-protein interactions. , 2000, Methods in enzymology.

[9]  T R Hughes,et al.  Genetic selection of peptide inhibitors of biological pathways. , 1999, Science.

[10]  D. Engelke,et al.  Yeast expression vectors using RNA polymerase III promoters. , 1994, Gene.

[11]  L. J. Maher,et al.  Combinatorial selection of a small RNA that induces amplification of IncFII plasmids in Escherichia coli. , 1998, Journal of molecular biology.

[12]  G. Soukup,et al.  Selection and characterization of RNAs that relieve transcriptional interference in Escherichia coli. , 1998, Nucleic acids research.

[13]  A. Colman-Lerner,et al.  "Mutagenesis" by peptide aptamers identifies genetic network members and pathway connections. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  M. Ptashne,et al.  Transcriptional activation by recruitment , 1997, Nature.

[15]  D J Segal,et al.  Toward controlling gene expression at will: specific regulation of the erbB-2/HER-2 promoter by using polydactyl zinc finger proteins constructed from modular building blocks. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[16]  L. Lim,et al.  An Abundant Class of Tiny RNAs with Probable Regulatory Roles in Caenorhabditis elegans , 2001, Science.

[17]  Jennifer A. Doudna,et al.  The chemical repertoire of natural ribozymes , 2002, Nature.

[18]  Neil J McKenna,et al.  A Steroid Receptor Coactivator, SRA, Functions as an RNA and Is Present in an SRC-1 Complex , 1999, Cell.

[19]  R. Knight,et al.  Analyzing partially randomized nucleic acid pools: straight dope on doping. , 2003, Nucleic acids research.

[20]  L. Hartwell,et al.  AKR1 encodes a candidate effector of the G beta gamma complex in the Saccharomyces cerevisiae pheromone response pathway and contributes to control of both cell shape and signal transduction , 1996, Molecular and cellular biology.

[21]  Thomas A. Steitz,et al.  The involvement of RNA in ribosome function , 2002, Nature.

[22]  R. Lanz,et al.  Distinct RNA motifs are important for coactivation of steroid hormone receptors by steroid receptor RNA activator (SRA) , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Wedel,et al.  Fishing the best pool for novel ribozymes. , 1996, Trends in biotechnology.

[24]  J. Sabina,et al.  Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. , 1999, Journal of molecular biology.

[25]  K. Struhl,et al.  Functional dissection of the yeast Cyc8–Tupl transcriptional co-repressor complex , 1994, Nature.

[26]  J. Mekalanos,et al.  Isolation of peptide aptamers that inhibit intracellular processes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Anthony D. Keefe,et al.  Functional proteins from a random-sequence library , 2001, Nature.

[28]  R. GeyerC,et al.  ペプチドアプタマーによる”変異原性”は遺伝的ネットワークメンバーと経路連結性を特定する , 1999 .

[29]  R R Breaker,et al.  Allosteric nucleic acid catalysts. , 2000, Current opinion in structural biology.

[30]  Jun Ma,et al.  A new class of yeast transcriptional activators , 1987, Cell.

[31]  R. Müller,et al.  Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. , 1995, Gene.

[32]  L James Maher,et al.  In vivo selection of spectinomycin-binding RNAs. , 2002, Nucleic acids research.

[33]  M. Ptashne,et al.  RNA sequences that work as transcriptional activating regions. , 2003, Nucleic acids research.