Award details

The CMR complex for prokaryotic RNA silencing

Reference	BB/K000314/1
Principal Investigator / Supervisor	Prof. Malcolm White
Co-Investigators / Co-Supervisors
Institution	University of St Andrews
Department	Biology
Funding type	Research
Value (£)	407,593
Status	Completed
Type	Research Grant
Start date	01/09/2012
End date	31/10/2015
Duration	38 months

Abstract

CRISPR (clusters of regularly interspaced palindromic repeats) is a recently discovered prokaryotic antiviral defence system. CRISPR loci in the genome store a record of past viral infection and act as an immune system, guiding cellular defence machinery to detect and degrade invading viral DNA or RNA. CRISPR associated (CAS) proteins form a variety of molecular machines, typified by the CASCADE complex that targets phage DNA and the CMR complex targetting viral RNA. Our laboratory was the first to purify the 400 kDa CMR complex (from Sulfolobus solfataricus) to homogeneity in significant amounts. This allowed us to initiate structural studies and begin to characterise the activities and mechanism of the complex. The viral RNA targeting activity of the CMR complex uses CRISPR RNA (crRNA) as a guide to target and degrade homologous RNA sequences in vitro. The reaction is sequence dependent and can result in cleavage of both the target and the guide RNA. This work was recently published in Molecular Cell. We now wish to characterise the mechanism of the reaction in detail, defining the active site(s) in the 7 subunits of the CMR complex, investigating the role of ATP in the reaction and determining the path of the bound crRNA and target RNA in the complex. Complimentary EM studies have recently been funded in collaboration with the Spagnolo lab in Edinburgh. In addition to the fundamental scientific interest in this complex process, we aim to harness the CMR complex to develop an RNA silencing system for prokaryotes analogous to that of RNAi in eukaryotes. To this end, we have identified a CMR system in a mesophilic bacterium and aim to transplant this operon into E. coli. Systematic analyses of the bacterial CMR complex will allow its key properties to be defined and a robust gene silencing system to be developed. The two main aims of the project can proceed independently but will reinforce one another.

Summary

The battle between viruses and cells is brutal and often compared to an "Arms Race". It is the most ancient of wars - as old as life itself. Viral infections cause millions of human casualties globally every year. The extreme evolutionary pressure exerted on cells by viruses, and vice versa, has been and remains a key driving force in evolution. For humankind, there are clear imperatives to explore the possibilities of virus-mediated cell killing for pathogenic bacteria and, conversely, to harness bacterial virus immunity systems in nature to the benefit of food production. Arguably, most exciting discovery in prokaryotic molecular biology in the past 5-10 years is the CRISPR system. CRISPRs are DNA sequences found in the genomes of many bacteria and archaea. They consist of short repeats flanking "spacers" that are often derived from viral sequences. The CRISPR region of the genome is transcribed to make an RNA copy, which is then chopped up to generate individual CRISPR RNA molecules, each with the potential to match the sequence of an invading virus. CRISPR-mediated viral defence (or Interference) is mediated by two large, complex molecular machines, CASCADE and CMR, targetting viral DNA and RNA, respectively. This project is focused on the CMR complex, which is less well understood. We have shown that CMR uses CRISPR RNA to degrade viral RNA targets in a sequence specific reaction. We now wish to understand the molecular basis for this reaction, which is probably unique in prokaryotes. Studies will focus on the active sites of the enzyme that degrades the RNA and the details of the interactions between RNA and protein. Further, we aim to harness the CMR complex to develop sequence dependent RNA silencing in prokaryotes. This technology is not available in a robust form for prokaryotes, unlike in eukaryotes where the RNAi system is used to silence specific genes. If we can develop this technology it will open the door to new possibilities in the manipulation of gene expression in bacteria and archaea with many downstream applications.

Impact Summary

Academic impact Scientists working in academia and industry will benefit from the basic advances in our understanding of the mechanism of CMR-mediated RNA silencing. Development of a prokaryotic gene silencing technology could significantly enhance their ability to investigate gene function in a variety of prokaryotes. Outreach The public, particularly young people, will benefit from outreach activities linked to this application. The PI is committed to activities supporting the public understanding of science - evidenced by a track record of engagement with Schools, public lectures and science festivals such as the Cheltenham science festival. School pupils will also get an opportunity to attend the laboratory and gain work experience (average of 1 student per year in the last 3 years). The team employed on the grant undertake to prepare and deliver an exhibit for science festivals (funds are requested for this). The subject - the battle between viruses and cells and its role in evolution - lends itself to public engagement opportunities. This work will expose the public, particularly young people, to exciting science and enhance their understanding of evolution. Research and professional skills The project will support two key members of staff in St Andrews. Dr Jing Zhang will have the opportunity to capitalise on her excellent work on the CMR system and obtain a number of high impact papers that will enhance her opportunities for career development. Dr Graham is pursuing a career as a research technician and has received 50% BBSRC funding from a related grant. This grant will secure the balance of her funding for 3 years. Both researchers will have access to the award winning "Gradskills" courses run by the University of St Andrews, which aim to provide a wide variety of life skills. The grant, if funded, will also underpin the research of two PhD students in the White laboratory, one BBSRC funded, both working on the CRISPR system. Economic and Societal Impact Industry, including biotechnology and fermentation companies, stand to benefit from the knowledge and technology that will result from this project. In particular, the development of a targetted gene silencing technology could have many applications in a range of industries reliant on micro-organisms. Engineered resistance to phage could be a boon for the dairy and other fermentation industries. In addition, there could be long term consequences for health care in the area of "phage therapy", which represents an attractive alternative to antibiotic use at a time when microbial drug resistance is becoming an increasingly serious problem. A detailed understanding of the CRISPR system is highly desirable for efforts to develop tailored phage therapies.

Committee	Research Committee C (Genes, development and STEM approaches to biology)
Research Topics	Microbiology, Structural Biology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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