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

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laboratoriesProf. Dr. Chakrabarti

Sutapa Chakrabarti Center
Freie Universität Berlin/ Institute of Chemistry and Biochemistry

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Room 129 - Takustr. 6, 14195 Berlin

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+493083875094?


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Structural biochemistry of factors involved in mRNA degradation

Lab InfoPROJECT INFORMATION :

This research proposal aims to understand the molecular mechanisms and regulation of ARE-mediated mRNA decay driven by the protein Tristetraprolin (TTP), using structural (X-ray crystallography) and biochemical tools. The degradation of mRNA is an important step in post-transcriptional gene regulation as it serves to regulate not only the level, but also the quality, of gene expression. Highly labile mRNAs, which have very short half-lives in the cell, often contain AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) and are targeted for decay through the AMD pathway. As such, the AMD pathway and the proteins involved therein are implicated in several inflammatory and autoimmune disorders and form an important platform for therapeutic intervention. A well-studied example of a protein involved in AMD is TTP. TTP binds the ARE-sequences on its target transcripts and recruits mRNA degradation factors such as the CCR4-NOT deadenylation complex and the decapping enzyme, DCP2, to mediate mRNA decay. However, not all ARE-binding proteins facilitate mRNA degradation; some proteins such as HuR bind to ARE-sequences and stabilize the mRNA. Interesting, HuR and TTP have an overlapping RNA target pool in the cell. Therefore, the ability of TTP to mediate mRNA decay depends not only on different mRNA degradation factors it recruits but also on other ARE-binding proteins, particularly the ones that exert an opposite effect on the fate of the mRNA transcript. Although recruitment of the CCR4-NOT complex by TTP is well characterized, not much is known about how TTP engages DCP2 to cause mRNA decay. Furthermore, it is not clear if TTP and HuR mediate their antagonistic effects only through a competition in binding the target mRNA (where HuR displaces TTP from the mRNA to block degradation) or if HuR is actively involved in blocking assembly of the mRNA degradation factors on the TTP-targeted mRNA. To this end, we propose to use structural and biochemical tools to probe the interaction of TTP with DCP2 to get a comprehensive picture on how different mRNA degradation factors are recruited by TTP. Furthermore, we wish to investigate the composition of the decapping-competent mRNP complex that is assembled in cells upon recruitment of DCP2 to a TTP-mRNA and determine how this composition/assembly is altered in the presence of HuR. We believe that the proposed experiments will provide us deep insight into the dynamics of mRNP assembly and remodelling that goes hand in hand with mediating and regulating TTP-dependent mRNA degradation. The proposed research will also be a distinct step towards understanding the role of ARE-binding proteins in context of their functional mRNPs and will enable us to better elucidate the dynamic interactions and molecular mechanisms that dictate ARE-mediated decay.




Lab techsKEY TECHNOLOGIES :

- X-ray crystallography
- In vitro tools for analyzing protein-protein/protein-RNA interactions




PublicationsPUBLICATIONS :

Chakrabarti, S., Bonneau, F., Schüssler, S. Eppinger, E. and Conti, E. (2014) Phosphodependent and phospho-independent recognition of the helicase UPF1 by the NMD factors SMG5-SMG7 and SMG6. Nucleic Acids Res. 42(14): 9447-60

Chakrabarti, S., Jayachandran, U., Bonneau, F., Fiorini, F., Basquin, C., Domcke, S., Le Hir, H. and Conti, E. (2011) Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2. Mol Cell, 41(6): 693-703

Chawla A., Chakrabarti S., Ghosh, G. and Niwa, M. (2011) Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase. J. Cell Biol., 193(1): 41-50

Ngo, J.C., Giang, K., Chakrabarti, S., Cheng-Ting, M., Huynh, N., Hagopian, J.C., Dorrestein, P.C., Fu, X.D., Adams, J.A. and Ghosh, G. (2008) A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1. Mol Cell, 29(5): 563-76

Ngo, J.C.*, Chakrabarti, S.*, Ding J.H., Velzques-Dones, A., Nolen, B., Aubol, B.E., Adams, J.A., Fu, X.D. and Ghosh, G. (2005) Interplay between SRPK and Clk/Sty kinases in phosphorylation of the splicing factor ASF/SF2 is regulated by a docking motif in ASF/SF2. Mol Cell, 20(1): 77-89