Prof. Dr. Niessing
Ludwig-Maximilians-Universität München, Biomedical Center (BMC) Department of Cell Biology
Großhaderner Str. 9 82152 Planegg-Martinsried
Send an Email
Colaboration with Prof. Dr. Vigo Heissmeyer
PROJECT INFORMATION :
The inducible costimulator of T cells (Icos) is critical for humoral immune responses since its loss- or gain-of-function is linked to immunodeficiency or autoimmunity, respectively. Icos has been shown to be under extensive post-transcriptional control. The published information so far demonstrates the regulation of Icos mRNA by the RNA-binding protein Roquin, the endonuclease Regnase-1 as well as several miRNAs. Furthermore, our preliminary data identified a novel cofactor in Roquin-mediated Icos repression.
The current knowledge typically relates the identification of one cis-element within an mRNA 3’ UTR to the regulation by one trans-acting factor. However, these noncoding sequences can be very long and often contain many different binding sites for trans-acting factors that either work independently, cooperatively or even interfere with each other’s function. In addition, there is little information how distant sequences or secondary structures influence the folding or accessibility of each other and jointly contribute to the post-transcriptional regulation of an entire mRNA.
Here, we focus on the long 3’ UTR of the mouse Icos mRNA for which we propose to acquire complementary information from several unbiased approaches leading to in depth structure-function studies that will then elucidate its combinatorial regulation. First, we will identify the proteome that is associated with this mRNA after cross-linking and sequence-specific pull-down of the mRNA from cell extracts. We will then analyze the secondary structure of the encoded sequence of the Icos 3’ UTR by chemical probing. Third we will systematically address the functional importance of sequences in this 3’ UTR by comprehensive random mutagenesis with about 11,000 sequence alterations. This, to our knowledge unprecedented approach, will systematically test individual mutations in the physiologic molecular context in primary T cell cultures to identify all regulatory elements of this long 3’ UTR. Finally, we shall bring together all the information and ask whether the individual trans-acting factors bind their cis-elements independently or in cooperation, how they interact with their cofactors, the translation machinery and factors of mRNA decay.
Overall, this work shall establish the functional and structural landscape of the Icos mRNA cis-elements that is then accessed by combinations of trans-acting factors, which engage in different physical and functional co-operations to realize regulation of the Icos mRNA in a context-dependent, differential or dynamic manner.
Focus of the group:
Post-transcriptional gene regulation and structure-based drug discovery
KEY TECHNOLOGIES :
- X-ray crystallography
- Protein and RNA biochemistry
- Chemical probing of RNA structures
- Quantitative interaction studies (ITC, SPR etc)
Janowski R., Heinz G.A., Schlundt A., Gruber A.R., Blank M., Buhmann R., Zavolan M., Niessing D.#, Heissmeyer V.#, Sattler M.#: Roquin recognizes a non-canonical hexa-loop structure in the 3’ UTR of Ox40.
Nature Communications 7,11032 (2016). (# Shared corresponding authorship)
Weber J., Bao H., Hartlmüller C., Wang Z., Windhager A., Janowski R., Madl T., Jin P., Niessing D.: Structural basis of nucleic-acid recognition and double-strand unwinding by the essential neuronal protein Pur-alpha.
eLIFE 5, pii: e11297 (2016).
Schlund A., Heinz G.A., Janowski R., Geerlof A., Stehle R., Heissmeyer V.#, Niessing D.#, Sattler M.#: Structural basis for RNA recognition in Roquin-mediated post-transcriptional gene regulation
Nature Struct. Mol. Biol. 21(8), 671-678 (2014). (# Shared corresponding authorship) Covered by Faculty 1000.
Heym, R.H., Zimmermann, D., Oekten, Z., Kovar, D., Niessing, D.: In vitro reconstitution of an mRNA-transport complex reveals mechanisms of assembly and motor activation.
J. Cell Biol. 203(6), 971-984 (2013).
Müller M., Heym R., Mayer, A., Kramer, K., Schmid, M, Urlaub, H., Jansen, R.P., Niessing, D.: A Cytoplasmic Complex Mediates Specific mRNA Recognition and Localization in Yeast.
PLoS Biology 9 (4), e1000611 (2011).