Identification and characterization of RNA guanine-quadruplex binding proteins.
Guanine quadruplex (G-quadruplex) motifs in the 5' untranslated region (5'-UTR) of mRNAs were recently shown to influence the efficiency of translation. In the present study, we investigate the interaction between cellular proteins and the G-quadruplexes located in two mRNAs (MMP16 and ARPC2). Formation of the G-quadruplexes was confirmed by biophysical characterization and the inhibitory activity on translation was shown by luciferase reporter assays. In experiments with whole cell extracts from different eukaryotic cell lines, G-quadruplex-binding proteins were isolated by pull-down assays and subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The binding partners of the RNA G-quadruplexes we discovered included several heterogeneous nuclear ribonucleoproteins, ribosomal proteins, and splicing factors, as well as other proteins that have previously not been described to interact with nucleic acids. While most of the proteins were specific for either of the investigated G-quadruplexes, some of them bound to both motifs. Selected candidate proteins were subsequently produced by recombinant expression and dissociation constants for the interaction between the proteins and RNA G-quadruplexes in the low nanomolar range were determined by surface plasmon resonance spectroscopy. The present study may thus help to increase our understanding of the mechanisms by which G-quadruplexes regulate translation.
Institute of Biotechnology, Department of Applied Biochemistry, TUB 4/3-2, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.
Annekathrin
A
von Hacht
Institute for Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Oliver
O
Seifert
RiNA GmbH, Volmerstraße 9, 12489 Berlin, Germany.
Marcus
M
Menger
Institute of Biotechnology, Department of Applied Biochemistry, TUB 4/3-2, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.
Tatjana
T
Schütze
Institute for Molecular Biosciences, University of Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt/Main, Germany.
Amit
A
Arora
Max Planck Institute for Molecular Genetics, Ihnestraße 63-73, 14195 Berlin, Germany Max Planck Institute of Colloids and Interfaces, Am Mühlenberg 1, 14476 Potsdam, Germany.
Zoltán
Z
Konthur
Institute of Biotechnology, Department of Bioprocess Engineering, ACK-24, Technische Universität Berlin, Ackerstraße 76, 13355 Berlin, Germany.
Peter
P
Neubauer
Institute of Biotechnology, Department of Applied Biochemistry, TUB 4/3-2, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany.
Anke
A
Wagner
Institute of Chemistry and Biochemistry, Freie Universität Berlin, Thielallee 63, 14195 Berlin, Germany.
Christoph
C
Weise
Institute of Biotechnology, Department of Applied Biochemistry, TUB 4/3-2, Technische Universität Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany jens.kurreck@tu-berlin.de.
Jens
J
Kurreck
D020121:5' Untranslated Regions; D051377:Actin-Related Protein 2 / Q000235:genetics* / Q000378:metabolism; D054856:G-Quadruplexes; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D053513:Matrix Metalloproteinase 16 / Q000235:genetics* / Q000378:metabolism; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism*
D020121:5' Untranslated Regions; D051377:Actin-Related Protein 2; D016601:RNA-Binding Proteins; D053513:Matrix Metalloproteinase 16