A structural and biochemical approach to investigating the composition, architecture and functionality of UPF1-centric degradosomes assembled during turnover of functional mRNA in eukaryotes.

• Applicant: Professorin Sutapa Chakrabarti, Ph.D.
• Subject Area: Structural Biology ; General Genetics and Functional Genome Biology ; Biochemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 463158693

This research proposal aims to investigate the composition, architecture and function of multi-protein/RNA complexes or degradosomes assembled by the RNA helicase UPF1 in pathways of functional mRNA turnover, using a combination of biochemical, structural and cell-based methods. The decay of mRNA is a crucial step in post-transcriptional gene regulation; its mis-regulation typically leads to cellular pathologies. Not surprisingly therefore, cells have devised mechanisms to make the process of mRNA decay as efficient and robust as possible. One of the ways by which cells streamline the decay of functional mRNA is by assembling decay-competent RNA-protein complexes known as degradosomes. The minimal degradosome contains an RNA helicase and a ribonuclease, while more complex assemblies might include a polymerase and a “sensor” protein that signals for RNA decay. The helicase UPF1, best known for its function in nonsense-mediated mRNA decay (NMD), is also involved in several pathways that mediate turnover of functional cellular mRNA. While the molecular mechanisms of UPF1 in context of an NMD-assembly are very well understood, comparatively little is known about its recruitment and functionality in pathways of mRNA turnover. To this end, I propose to investigate two UPF1-dependent mRNA turnover pathways – Staufen-mediated mRNA decay (SMD) and decay of replication-dependent histone mRNAs. Previous work suggested that UPF1 is recruited to these pathways by direct interactions with specific trans-acting protein factors, namely Staufen in the SMD pathway and the stem-loop binding protein (SLBP) in histone mRNA decay. UPF1 unwinds the structured RNA and, as a result, facilitates decay of the target mRNA transcript. Our recent studies showed, for the first time, that in addition to UPF1, the NMD factor UPF2 plays a crucial role in SMD by forming a scaffold for assembling a decay-competent mRNA-protein complex (mRNP). The involvement of different trans-acting factors in SMD and histone mRNA decay points to the assembly of a distinct decay-inducing mRNP in each case. Since assembly of a UPF1-mRNP is the trigger for initiating mRNA decay both in SMD and in histone mRNA decay, it is imperative to investigate this process at a molecular level. Therefore, we propose to determine the composition of the UPF1-centric degradosomes in the two decay pathways, and investigate their step-wise assembly and disassembly and the molecular mechanisms that lead to target mRNA decay in each case. We will employ a combination of structural (X-ray crystallography and cryo-electron microscopy) and biochemical methods, together with cell-based tools to address these aspects of mRNA decay. In addition to deciphering the mechanisms of these important cellular decay pathways, our studies will also provide insight into the crosstalk among different mRNA degradation pathways and will bring us closer towards defining the principles of mRNP/degradosome assembly in eukaryotes.

• DFG Programme: Research Grants