DFG Research Group FOR855

    Experimental identification of mammalian mRNAs that are regulated by microRNAs

    Prof. Dr. Gunter Meister

    Project Summary:

    MicroRNAs (miRNAs) form an evolutionary conserved class of small non-coding RNAs that repress gene expression by destabilizing specific mRNAs or inhibiting mRNA translation without affecting its stability. Although many protein components of the miRNA pathway have been discovered and their mode of action is becoming more and more unraveled, only very little is known about the mRNAs that are regulated by miRNAs. Such target mRNAs have been predicted bioinformatically but only a few of them have been experimentally validated. Due to the vast amount of potential target mRNAs a systematic validation is hardly feasible. Strategies that aim for the experimental identification of such miRNA target mRNAs from specific cells and tissues will help to overcome such difficulties. We therefore proposed the development of an experimental strategy to isolate and identify mRNA targets that are specific to one given miRNA. We will fuse a biotin-tag to the 3’ end of a synthetic miRNA and transfect it into different cell lines. RNA-protein complexes containing the respective biotinylated miRNAs will be isolated using streptavidin-coated beads. Purified mRNAs will be cloned and sequenced. In further experiments the identified mRNAs will be validated using reporter systems. Finally, the biological roles of the miRNA-mRNA interactions will be investigated.

    Selected publications (2007-09):

    1.    Zhu J.Y., Pfuhl T., Motsch N., Barth S., Nicholls J., Grässer F. & Meister G. (2009) Identification of novel Epstein-Barr Virus microRNA genes from Nasopharyngeal Carcinomas. Journal of Virology, 83(7):3333-41.

    2.    Weinmann, L., Höck, J., Ivacevic T., Ohrt T., Mütze J., Schwille P., Kremmer E., Benes V., Urlaub, H., & Meister G. (2009) Importin 8 is a gene silencing factor that targets Argonaute proteins to distinct mRNAs. Cell, 136, 496-507.

    3.    Ender C., Krek A., Friedländer M.R., Beitzinger M., Weinmann L., Chen W., Pfeffer S., Rajewsky N. & Meister G. (2008) A human snoRNA with microRNA-like functions. Molecular Cell, 32, 519-528.

    4.    Ohrt T., Mütze J., Staroske W., Weinmann L., Höck J., Crell K., Meister G. & Schwille P. (2008) Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells. Nucleic Acids Research, 36(20):6439-49.

    5.    Lindner B., Plöttner O., Kroiss M., Hartmann E., Laggerbauer B., Meister, G., Keidel E. & Fischer U. (2008) Tdrd3 is a novel stress granule-associated protein interacting with the Fragile-X syndrome protein FMRP. Human Molecular Genetics, 17, No. 20, 3236-3246.

    6.    Rüdel S., Flatley A., Weinmann L., Kremmer E. & Meister G. (2008) A multifunctional human Argonaute2-specific monoclonal antibody. RNA, 14, 1244-1253.

    7.    Chen P. Y., Weinmann L., Gaidatzis D., Pei Y., Zavolan M., Tuschl T. & Meister G. (2007) Strand-specific 5’-O-methylation of siRNA duplexes controls guide strand selection and targeting specificity. RNA, 14, 1-12.

    8.    Höck J., Peters L., Ender C., Rüdel S., Kremmer E., Urlaub H. & Meister G. (2007) Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells. EMBO Reports, 8, 11, 1052-1060.

    9.    Barth S., Pfuhl T., Mamiani A., Ehses C., Roemer K., Kremmer E., Jäker C., Höck J., Meister G. & Grässer F. (2008) Epstein-Barr Virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. Nucleic Acids Research, 36(2), 666-675.

    10.    Beitzinger M., Peters L., Zhu J. Y., Kremmer E. & Meister G. (2007) Identification of microRNA targets from isolated Argonaute protein complexes. RNA Biology, 4(2).

    11.    Tarasov V., Jung P., Verdoodt B., Lodygin D., Epanchintsev A., Menssen A., Meister G. & Hermeking H. (2007) Differential regulation of microRNAs by p53 revealed by massively parallel sequencing: miR-34a is a p53 target that induces apoptosis and G(1)-arrest. Cell Cycle, 6(13), 1586-1593.