Unbiased selection of localization elements reveals cis-acting determinants of mRNA bud localization in Saccharomyces cerevisiae.

Cytoplasmic mRNA localization is a mechanism used by many organisms to generate asymmetry and sequester protein activity. In the yeast Saccharomyces cerevisiae, mRNA transport to bud tips of dividing cells is mediated by the binding of She2p, She3p, and Myo4p to coding regions of the RNA. To date, 24 bud-localized mRNAs have been identified, yet the RNA determinants that mediate localization remain poorly understood. Here, we used nonhomologous random recombination to generate libraries of sequences that could be selected for their ability to bind She-complex proteins, thereby providing an unbiased approach for minimizing and mapping localization elements in several transported RNAs. Analysis of the derived sequences and predicted secondary structures revealed short sequence motifs that mediate binding to the She complex and RNA localization to the bud tip in vivo. A predicted single-stranded core CG dinucleotide appears to be an important component of the RNA-protein interface, although other nucleotides contribute in a context-dependent manner. Our findings further our understanding of RNA recognition by the She complex, and the methods used here should be applicable for elucidating minimal RNA motifs involved in many other types of interactions.

[1]  E. Mugumya.  Small families mean better health for mothers and kids. , 1994, Africa women & health : a Safe Motherhood magazine.

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

[3]  Marvin Wickens,et al.  RNA-protein interactions in the yeast three-hybrid system: affinity, sensitivity, and enhanced library screening. , 2005, RNA.

[4]  P. Gendron,et al.  Identification of a Conserved RNA Motif Essential for She2p Recognition and mRNA Localization to the Yeast Bud , 2005, Molecular and Cellular Biology.

[5]  R. Singer,et al.  RNA localization: different zipcodes, same postman? , 1998, Trends in cell biology.

[6]  R. Singer,et al.  She2p is a novel RNA‐binding protein that recruits the Myo4p–She3p complex to ASH1 mRNA , 2000, The EMBO journal.

[7]  J. L. Smith,et al.  Multiple RNA regulatory elements mediate distinct steps in localization of oskar mRNA. , 1993, Development.

[8]  David R. Liu,et al.  Nucleic acid evolution and minimization by nonhomologous random recombination , 2002, Nature Biotechnology.

[9]  R. Singer,et al.  Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo , 1999, Current Biology.

[10]  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.

[11]  P. Brown,et al.  Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts using DNA microarray analysis , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[12]  A. Mccarthy.  Development , 1996, Current Opinion in Neurobiology.

[13]  R. Jansen,et al.  She2p, a novel RNA‐binding protein tethers ASH1 mRNA to the Myo4p myosin motor via She3p , 2000, The EMBO journal.

[14]  T. D. Schneider,et al.  Sequence logos: a new way to display consensus sequences. , 1990, Nucleic acids research.

[15]  J. Ellenberg,et al.  Ribonucleoprotein-dependent localization of the yeast class V myosin Myo4p , 2002, The Journal of cell biology.

[16]  R. Vale,et al.  The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Frith,et al.  A Ubiquitous and Conserved Signal for RNA Localization in Chordates , 2002, Current Biology.

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

[19]  R. D. Gietz,et al.  Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. , 2002, Methods in enzymology.

[20]  E. R. Gavis,et al.  Overlapping but distinct RNA elements control repression and activation of nanos translation. , 2000, Molecular cell.

[21]  P. Philippsen,et al.  Additional modules for versatile and economical PCR‐based gene deletion and modification in Saccharomyces cerevisiae , 1998, Yeast.

[22]  C. Cote,et al.  Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes. , 1997, Development.

[23]  G. Crooks,et al.  WebLogo: a sequence logo generator. , 2004, Genome research.

[24]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[25]  Kim Nasmyth,et al.  ASH1 mRNA localization in yeast involves multiple secondary structural elementsand Ash1 protein translation , 1999, Current Biology.

[26]  P. Macdonald,et al.  Mutational Analysis of an RNA Recognition Element That Mediates Localization of bicoid mRNA , 1998, Molecular and Cellular Biology.