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

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laboratoriesProf. Dr. Sattler

Michael Sattler Center
Technical University of Munich and Helmholtz Center

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Lichtenbergstrasse 4, 85747 Garching

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00498928913418


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Structural biology of RNPs

Colaboration with Dr. Julian König



Lab InfoPROJECT INFORMATION :

RNA-binding proteins (RBPs) are critical players in the posttranscriptional control of gene expression and regulate various mRNA processing steps, including splicing, 3’ end processing and translation. Most often, multiple RBPs come together to form large messenger ribonucleoprotein particles (mRNPs), and the cooperative action of these mRNPs controls the fate and function of each transcript. However, the molecular features that define this so-called mRNP code in a given functional context remain poorly understood.
Here, we propose to combine transcriptome-wide approaches using in vitro and in vivo iCLIP with biophysical and structural studies to unravel the molecular mechanisms of mRNP assembly at cis elements in the 3’ splice site of human introns. A pioneering event in splice-site recognition is binding of the U2 auxiliary factor (U2AF) heterodimer with its subunits U2AF65 and U2AF35 to the polypyrimidine (Py)-tract and the invariant 3’ splice site AG motif (3’ AG), respectively. Intriguingly, the Py-tracts in human introns exhibit large sequence variations, but how these variations influence an intron’s splicing competence remains poorly understood. Py-tract recognition by U2AF65 is frequently modulated by other RBPs, including U2AF35 as well as splicing factor 1 (SF1), which binds to the upstream branch point sequence (BPS). Moreover, splicing fidelity is enhanced by proof-reading against binding of U2AF to cryptic Py-tracts without 3’ AG. Understanding the molecular mechanisms of 3’ splice-site recognition and specifically the role of U2AF35 is of high importance, since mutations in this RBP are associated with human disease.

The goal of this project is to dissect the dynamic interplay of RBPs at the 3’ splice site. In particular, we will address the following key questions: 1) What are the rules of U2AF binding at the highly diverse 3' splice sites in human introns? What is the impact of U2AF35 and its disease-linked mutations on mRNP assembly? 2) What is the contribution of SF1 binding and branch point recognition to U2AF recruitment during mRNP assembly? What renders a 3' splice site sensitive to the presence of SF1 and how is this modulated by SF1 phosphorylation? 3) What are the molecular mechanisms of proof-reading and the removal of erroneously assembled mRNPs at 3' splice sites?

Bridging the fields of structural biology and functional genomics, our approach will help to decipher the mRNP code of 3’ splice-site recognition and splicing regulation - from the mechanistic principles of combinatorial and dynamic RBP binding at the molecular level to the functional consequences of mRNP assembly in living cells. Beyond splice-site recognition, our results will offer a blueprint for understanding the assembly of other mRNPs. Given the unique combination of complementary methods employed, we expect numerous interactions with other researchers within the SPP1935.



Lab techsKEY TECHNOLOGIES :

- NMR-spectroscopy
- SAXS
- SANS
- Crystallography
- ITC
- Integrated structural biology



PublicationsPUBLICATIONS :

Hennig J, Militti C, Popowicz GM, Wang I, Sonntag M, Geerlof A, Gabel F, Gebauer F, Sattler M
Structural basis for the assembly of the Sxl–UNR translation regulatory complex
(2014) Nature, 515:287-90.

Schlundt A, Heinz GA, Janowski R, Geerlof A, Stehle R, Heissmeyer V, Niessing D, and Sattler M. Structural basis for RNA recognition in roquin-mediated post-transcriptional gene regulation.
(2014) Nat Struct Mol Biol 21, 671-8.

Mackereth CD, Madl T, Bonnal S, Simon B, Zanier K, Gasch A, Rybin V, Valcarcel J, Sattler M. Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF
(2011) Nature 475, 408-11.

Tripsianes K, Madl T, Machyna M, Fessas D, Englbrecht C, Fischer U, Neugebauer KM, and Sattler M Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins. (2011) Nat Struct Mol Biol 18, 1414-20.

Liu Z, Luyten I, Bottomley MJ, Messias AC, Houngninou-Molango S, Sprangers R, Zanier K, Krämer A, and Sattler M Structural basis for recognition of the intron branch site RNA by splicing factor 1.
(2001) Science 294, 1098-102.