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Structural and biophysical mechanism of microRNA nuclear export

Final Report Summary - MICRORNA EXPORT (Structural and biophysical mechanism of microRNA nuclear export)

In eukaryotic organisms the genetic material that codes for protein is segregated from the protein factories, termed ribosomes, by the nuclear envelope. For protein to be produced the 'genetic code' must first be copied from the DNA to RNA, a complex process termed transcription. The RNA is then transported to the cytoplasm through the nuclear envelope, passing through large multi-protein tunnels termed nuclear pore complexes (NPCs). RNAs in the cytoplasm can be translated into proteins or become involved in the regulation of a host of cellular processes. The gene expression pathway consists of numerous steps that are tightly coupled and show a high degree of interdependence. Integration of the steps that lead from transcription to RNA nuclear export relies on a complex network of interactions between activated genes, processing factors and the NPC. For a subset of genes, this integration is achieved by tethering actively transcribed genes to the NPC, a process known as 'gene-gating'.

This project has focused upon understanding the biological process of gene-gating at the molecular level. We have investigated the structure and function of the small nuclear localised protein Sus1 in gene gating. Sus1 is a central component of two complexes that are involved in the gene expression pathway termed the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex and the transcription and export-2 (TREX-2) complex. The TREX-2 complex binds the mRNA export factors Mex67 and Mtr2 as well as NPCs, and by interacting with both SAGA and NPCs the TREX-2 complex facilitates gene-gating.

This award initially allowed me to conduct key experiments into the in vitro binding of Sus1 to the TREX-2 complex. These novel-binding assays facilitated the completion of a pre-existing project in the lab, which was subsequently published in Molecular Cell. Moreover, this provided a starting point for the major focus of this fellowship, specifically looking at the structure and function of Sus1 in the SAGA complex and the mechanism whereby Sus1 links both the SAGA and TREX-2 complexes and facilitates gene-gating. Results from this section of the project were published in the Journal of Biological Chemistry.

In this project we solved the crystal structure of the N-terminal region of Sgf11 bound to Sus1, its direct binding partner in the SAGA complex. We have shown that Sgf11 forms an extended helix around which Sus1 wraps in a manner that has some similarities to the way in which it binds to Sac3 in the TREX-2 complex. However, the Sus1-binding site on Sgf11 is somewhat shorter than on Sac3 and is based on narrower hydrophobic stripe. We engineered mutants based on the structure of the complex that disrupt the Sgf11-Sus1 interaction and have used these to confirm the importance of the hydrophobic stripe in molecular recognition.

It was initially suggested that Sus1 could function to physically bridge the SAGA and TREX-2 complexes directly. As such, Sus1 was proposed to be the structural and functional key to the process of gene-gating. The structural and biological data derived from this project however have led to a model in which Sus1 forms a component of separate sub-complexes in SAGA and TREX-2 rather than a single Sus1 chain linking the two complexes.

This award has also supported further efforts to ascertain the mechanism whereby the SAGA complex and the TREX-2 complexes are linked to facilitate gene-gating. Protein crystals have been obtained of other important regions of the TREX-2 complex that will provide further data on the key interactions of the TREX-2 complex during gene-gating. It is the aim to continue working on these structures and for these data to be incorporated with biological and functional studies for publication in a high-impact peer reviewed scientific journal. In conclusion, this award has facilitated a multi-faceted project that has investigated the mechanism of gene-gating, a process that is fundamental to the gene expression pathway.