The CMR complex for prokaryotic RNA silencing

Lead Research Organisation: University of St Andrews
Department Name: Biology


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.

Technical Summary

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.

Planned Impact

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.
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.
Description In the three years since this grant was funded there have been a series of important advances in our understanding of the CRISPR system arising from the work of many laboratories worldwide. 36 papers were published in 2009, 49 in 2010 and 82 in 2011. In brief, the first description of the machinery for viral DNA degradation, named CASCADE, arose from studies in E. coli (published in Science by the van der Oost lab in 2008). The corresponding machinery for viral RNA degradation in Pyrococcus furiosus, named the CMR complex (corresponding to the Polymerase Cassette Complex in our original application), was published by the Terns lab in Cell in 2009. The enzyme that generates CRISPR RNA (crRNA) (named pDicer in our original application), was identified as Cas6 in 2008 and several structures have since been reported.

In this fast moving field, we have begun to make a significant impact from the structural and functional studies of the CRISPR system in S. solfataricus, as follows:

1. Structure and function of archaeal CASCADE.
Systematic co-expression of CRISPR proteins (Objective 1b) led to the identification of a stable interaction between Cas5 and Cas7. We recognised this pair as the core of the CASCADE complex for viral DNA targetting in archaea. In collaboration with the Lawrence lab (Montana), we reported the first structure of the widely conserved Cas7 protein, revealing an RRM fold related to that found in other CAS proteins. This helped to simplify our understanding of the evolution of the CRISPR System. We showed that Cas7 has an important role in crRNA binding, probably forming the backbone of the CASCADE complex. Further, we identified a third interacting subunit of the complex, Csa5, and characterised the bound crRNA (Objective 2a). This work was published in JBC in 2011.
In further work thus far unpublished, we have solved the crystal structure of the Csa5 subunit of CASCADE, which adopts a helical complex structure that may be relevant to the overall structure of the complex. This structure will be published in 2012 along with biochemical evidence of archaeal CASCADE function. Structural studies of the archaeal CASCADE complex are ongoing.

2. Structure and mechanism of the CMR complex (Objective 2d).
CMR is the only known prokaryotic RNA silencing machinery and as such has been the subject of intense interest. The 7-subunit CMR complex for crRNA-mediated RNA degradation was purified to homogeneity from S. solfataricus. Tagged versions were constructed and expressed, allowing targetted mutation of subunits. In vitro, the complex had the expected RNA targetting activity but with a number of important differences from the previously published Pyrococcus complex. In particular, we demonstrated that the endonuclease activity was sequence dependent and that both target and guide RNA could be cleaved. Structural studies revealed the structure of the Cmr7 subunit and an EM reconstruction of the entire 400 kDa complex (collaboration with Laura Spagnolo, Edinburgh). This work was published in Molecular Cell (online 5th Jan 2012). The accompanying grant application describes the future work planned for this aspect of the CRISPR system. Structural studies will continue in work led by the Spagnolo lab and recently funded by BBSRC.

3. Insights from Deep sequencing of crRNA.
We have purified the RNA components of 3 different CRISPR protein complexes (CASCADE and two CMR complexes) and subjected this to deep sequencing. The data were obtained very recently and have not been analysed fully, but are already yielding new insights. We have noted big differences in the representation of adjacent CRISPR spacers that may relate to differential folding potential at the RNA level. This is being followed up with in vitro studies. Dramatic differences in the RNA content of the two CMR complexes are also apparent. One has a wide range of RNA bound, including sRNA, mRNA, rRNA and antisense RNA generated by transposons, suggesting alternate roles for this complex in RNA metabolism. In contrast, the second CMR complex appears to bind only crRNA, suggesting a specific mechanism for crRNA loading.

4. Role of Cas6 in crRNA generation (Objective 2b)
Cas6 is an independent enzyme that feeds crRNA to both the CASCADE and CMR complexes in archaea, unlike the situation in many bacteria where the equivalent enzyme is a subunit of the complex. Although the Cas6 structure from P. furiosus has been described and a mechanism proposed, significant questions remain about the activity of the enzyme and putative catalytic residues are not conserved. We have shown that S. solfataricus Cas6 cleaves crRNA in vitro and have recently solved the crystal structure of the enzyme, revealing a novel dimeric arrangement (Figure 1). Deep sequencing suggests differential processing of crRNA may be an important factor in the CRISPR system. We are currently investigating the catalytic mechanism of the Sulfolobus enzyme, investigation interactions with other CRISPR components and attempting to crystallise a co-crystal structure with RNA bound.

5. Initial studies of the Cas1-Cas2-Cas4 adaptation mechanism (Objective 2e).
The capture of new viral sequences (adaptation) is known to require Cas1 and Cas2 but the mechanism remains enigmatic. We have succeeded in expressing both these proteins and solved the crystal structure of Cas2. We have now shown that Cas2 is a metal independent ribonuclease (in contrast to the only published report which suggests a divalent metal is essential). We also have preliminary data suggesting that Cas1 acts as an endonuclease specific for supercoiled DNA, an important clue to function as the new DNA must be spliced into the host cell genome. Further, we have shown that the Cas4 protein is a nuclease with an iron-sulphur cluster, related to the AddAB nuclease (paper in preparation for publication in 2012). Future work will be aimed at the reconstitution of the adaptation machinery in vitro, allowing determination of the reaction mechanism in detail.
Exploitation Route CRISPR is a rapidly moving field and there have been many advances related to the work in this grant since the grant ended.
Sectors Agriculture, Food and Drink,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology