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Molecular mechanisms of mRNA decay

Final Report Summary - MRNA DECAY (Molecular mechanisms of mRNA decay)

The proper regulation of gene expression is fundamental for the homeostasis of all organisms. It is thus not surprising that multiple mechanisms regulate the flow of genetic information from the genome into functional protein complexes. One such mechanism is through modulation of messenger ribonucleic acid (mRNA) stability, where removal of an mRNA from the translational pool, results in a down regulation of protein synthesis. We are interested in understanding how a large number of enzymes and regulatory factors assemble into a cellular machine that is able to efficiently degrade these mRNAs.

Many of the components (enzymes and regulatory factors) have been identified. For some of these proteins, a three-dimensional (3D) structure is known. Here, we study how these building blocks interact with each other and how motions within the enzymes module control the enzymatic activity. Especially the latter is largely undetermined and molecular motions often remain undetected, despite their importance in catalysis.

After an mRNA is deadenylated, it can either be degraded in the 3' to 5' direction by the exosome complex, or, alternatively, the mRNA can interact with the LSm1-7 complex that then in turn recruits the decapping (Dcp2) enzyme resulting in the degradation of the mRNA body in the 5' to 3' direction.

The exosome complex is a large enzyme complex of which the static structure has been determined in detail. Here, we studied the archaea exosome complex and complemented the known static structure with information regarding protein motions. To that end we have used novel methods in nuclear magnetic resonance (NMR) spectroscopy that are able to detect and quantify motions in complexes over 150 kDa. Unexpectedly, we found that the exosome is highly dynamic in solution and that the identified motions correlate with activator protein interactions. Our data thus provides the first insights in previously undetected dynamic processes that are important for the 3' to 5' mRNA degradation process. It should be noted that the details regarding protein motions we obtained in our studies are normally only achieved for proteins an order of magnitude smaller.

The LSm1-7 complex, like the exosome complex, interacts with the deadenylated 3' end of the mRNA and initiates the 5' to 3' degradation of the transcript. The LSm1-7 complex contains seven different protein chains and has structurally not been studied in detail, due to this complexity. Here, we have determined the high-resolution three-dimensional structure of an assembly intermediate of the LSm1-7 complex that contains three LSm proteins. This LSm657 complex forms a hexameric ring that can open in solution in order to incorporate additional LSm proteins. To study the full LSm complex, we developed a novel NMR method (LEGO-NMR) that produces asymmetric protein complexes that are NMR active in only a subset of the subunits. This significantly simplifies NMR sample preparation and the resulting NMR spectra. For the LSm complexes that we applied this new methodology to, we were able to determine that the central pore of the complex is used to interact with substrate RNA. Our data thus provides the first insights into the initial processes that eventually result in mRNA degradation in the 5' to 3' direction.

The Dcp2 enzyme performs the removal of the 5' protecting cap structure, a central step of the mRNA degradation process. The activity of this enzyme is, and must be, tightly regulated and a large number of regulatory factors have been identified. These include the prime activator Dcp1 and enhancer of mRNA - decapping protein 3 (Edc3), Scd6 and the LSm1-7 complex. Here, we determined the structural basis for the interaction between the Dcp2 enzyme and the Edc3 protein. Interestingly, our data revealed a large network of interactions between the Edc3 protein and Dcp2 that could stimulate processing body formation. Our data also shows that changes in the internal motions of the Dcp1:Dcp2 complex are likely responsible for the modulation of catalytic activity. This, once more, sheds light on the importance of studies that aim at determining protein motions with atomic resolution.

In summary, our results reveal novel insights in how multiple proteins interact to form a functional mRNA degradation machine. We show that a close interplay between protein structure and protein motions is able to regulate catalytic activity. Our results also form a solid basis for future studies that will include additional components of the mRNA degradation machinery. Our methodological advances will also allow us to initiate studies that aim at reconstituting the complete mRNA degradation machinery in vivo. In the long run these experiments will be able to reproduce cellular behaviour in a well-defined setting. This approach will be useful for studies that interfere with the degradation process and that can thus be of pharmaceutical use.

Project website: http://www.eb.tuebingen.mpg.de/research/research-groups/remco-sprangers.html