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SPP1935 -- Deciphering the mRNP code :
RNA-bound Determinants of Post-transcriptional Gene Regulation

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laboratoriesProf. Dr. Meister

Gunter Meister Center
University of Regensburg, Biochemistry Center Regensburg (BZR)

Adress
Universitätsstrasse 31, 93053 Regensburg

Phone
09419432847


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beta-propeller domains as novel RNA-binding modules

Lab InfoPROJECT INFORMATION :

Various proteins have been found in mRNPs and many of them directly contact the mRNA. However, a large number of these factors do not contain known RBDs. We have recently shown that β-propeller domains can establish sequence-specific contacts with mRNA substrates and regulate their expression. However, it is not clear how many β-propeller domains can contact RNA in addition to their protein binding potential. The widespread WD40 domain, for example, which folds into β-propellers, has been associated with mRNA binding, but direct experimental proof is lacking and the molecular basis for binding specificity and efficiency is largely unknown. Thus, the objectives and aims of this proposal are:
We have selected eight WD40 domain-containing proteins that directly contact mRNAs in cross-linking experiments. We will generate tools such as expression systems, recombinant proteins, specific antibodies, siRNAs and CRISPR/Cas-mediated knock out cell lines, which will be important for a comprehensive analysis of these proteins in mRNP function.
We have established and modified an RNA ligand selection procedure termed bind-n-seq. Recombinant WD40 proteins will be used in bind-n-seq experiments and specific binding motifs will be identified.
In our lab, we have recently solved the co-structure of the NHL domain together with its bound RNA motif. WD40 domains or larger protein fragments will be crystallized in complex with the RNA motif identified in aim 2.
In RIP (RNA immunoprecipitation) experiments, endogenous target mRNAs will be isolated and binding motifs will be identified. In addition, CLIP experiments will be performed to identify bound targets as well as the actual binding sites on mRNAs.
To unravel the functional consequences of the WD40 domain proteins on their mRNA targets, we will deplete the proteins (RNAi, CRISPR/Cas) from suitable cell lines and analyze consequences on target mRNAs. Reporter gene assays will be used to identify individual functional steps in which these proteins are involved.

Focus of the group
Identification of mRNA-protein interactions, structural biology



Lab techsKEY TECHNOLOGIES :

Bind’n’seq
EMSA
CLIP experiments



PublicationsPUBLICATIONS :

Hasler D., Lehmann G., Murakawa Y., Klironomos F., Jakob L., Graesser F.A., Rajewsky N., Landthaler M. & Meister G. (2016) The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway, Molecular Cell, 7;63(1):110-24.

Loedige I., Jakob L., Treiber T., Ray D., Stotz M., Treiber N., Hennig J., Cook K.B., Morris Q., Hughes T.R., Engelmann J.C., Krahn M.P., Meister G. (2015) The Crystal Structure of the NHL Domain in Complex with RNA Reveals the Molecular Basis of Drosophila Brain-Tumor-Mediated Gene Regulation. Cell Rep. 2015 Nov 10;13(6):1206-20.

Hauptmann J., Schraivogel D., Bruckmann A., Manickavel S., Jakob L., Eichner N., Pfaff J., Urban M., Sprunck S., Hafner M., Tuschl T., Deutzmann R. & Meister G. (2015) Biochemical isolation of Argonaute protein complexes by Ago-APP. Proc. Natl. Acad. Sci. USA., 112(38):11841-5.

Loedige I., Stotz M., Qamar S., Kramer K., Hennig J., Schubert T., Löffler P., Längst G., Merkl R., Urlaub H. & Meister G. (2014) The NHL domain of BRAT is an RNA-binding domain that directly contacts the hunchback mRNA for regulation. Genes Dev., 28(7): 749-764.

Pfaff J., Hennig J., Herzog F., Aebersold R., Sattler M., Niessing D. & Meister G. (2013) Structural features of GW – Argonaute protein interactions. Proc. Natl. Acad. Sci. USA., 110(40):E3770-9.