How Are Cellular RNAs Targeted For Maturation And Degradation?

Summary
The central dogma of molecular biology states that DNA makes RNA makes protein. The process of transcription, whereby DNA is "copied" into mRNA (messenger RNA) is the intermediate step in the production of proteins. However, the majority of transcription that takes place in a cell results in the production of RNA molecules that do not, in fact, encode for proteins. These diverse RNA species fall into various categories which can be divided based upon their structure, function and the proteins with which they associate with. The multiple species of RNA carry out a plethora of functions in a cell from catalysing chemical reactions to controlling levels of protein expression. Many of these RNA species, including mRNA, are not transcribed in their final functional form, and are subject to processing events in order to achieve their mature state. Further, these events are tightly regulated to ensure that the production of the RNA is accurate. If a fault in their production occurs, the aberrant RNA specie is subject to degradation, so as to prevent defective machinery engaging in the cell. One key factor that is implicated in both the maturation and degradation of almost all species of RNA is the exosome complex, which has the ability to remove specific regions of the RNA during processing or digest the complete RNA during degradation. One of the great conundrums is how the exosome recognises and is targeted to its numerous different RNA species. This project will uncover how the different RNA species are recognised.

Technical Summary
The exosome complex is required for the maturation and degradation of almost all species of RNA in eukaryotes. However, the mechanism by which the exosome recognises the majority of its substrates remains ill-defined. We previously determined that the nuclear exosome is recruited to two of its major rRNA substrates via the adapter proteins Nop53 and Utp18. Further we could identify that the recruitment was mediated via the "arch domain" of the helicase co-factor of the nuclear exosome, Mtr4. Strikingly, the conserved arch domain has only been identified in exosome-associated helicases, suggesting that it plays an important role in substrate recognition. This project will explore the molecular mechanisms that govern the recruitment of the exosome complex to its plethora of RNA substrates. This is a fundamental question that has implications for many fields of eukaryotic biology. We will systematically analyse the two exosome-associated helicases in human cells, Mtr4 and Ski2, that function with the nuclear and cytoplasmic forms of the exosome respectively. We will identify and characterise novel adapter factors and the RNA substrates that they target to the nuclear and cytoplasmic exosome complexes. We will achieve this using a combination of quantitative mass spectrometry (SILAC), protein-RNA crosslinking (CRAC) and RNAseq analysis. Together this analysis of arch interacting factors will broaden our understanding of the nature of exosome targeting and provide a global view of the role played by helicase cofactors in exosome recruitment.

Impact Summary
The proposed work will address the fundamental biological question of how the exosome complex identifies its numerous different RNA substrates. I anticipate that this work will provide benefit to, and have impact on, four main groups.

1. Biotechnology industry As our work focuses on the mechanisms regulating processing and degradation of RNA in human cells, our findings can inform work in industrial biotechnology. This work would be especially relevant to those where efficient processing and stability of mRNA is crucial for their system of analysis/production e.g. in the production of antibodies or other therapeutic compounds in mammalian cell.

2. Clinicians and patients The fundamental role of the exosome is important for human health, as dysregulation or mutation of the human exosome is linked to autoimmune diseases, leukaemia and neurological conditions. The exosome is also a component of cellular anti-viral defence mechanisms, as it contributes to the degradation of viral RNA. The identification of factors that act to target the exosome and therefore constitute key regulatory components, may in the future help to rationalise clinical observation and better understand mechanistic details of clinically relevant exosome variants and provide novel targets for potential therapeutic intervention.

3. Researchers engaged on the project- This project offers the possibility to train and mentor a post-doctoral researcher (PDRA). The PDRA will work in an exciting field of research and will become an expert in cutting edge techniques, in addition to bioinformatics and data management skills. The projects they will perform have the possibility to result in high-impact publications. Further the researcher will be encouraged to supervise students and enroll in career development courses. Collectively, this will allow the researcher to be highly competitive for future employment in either the public or private sector.

4. The general public. I plan to communicate the basis and significance of the research that I am performing through a number of public engagement initiatives run by the University of Sheffield in addition to the preparation of press-releases upon publication of significant research findings.