The initiation of mRNA degradation by the direct entry of RNase E and the degradosome with implications for non-nucleolytic gene control

Many bacterial species pose a significant health-risk to humans, animals and plants, while others are an important source of nutrients and medicines including antimicrobials and anti-cancer agents. Some, including one called Escherichia coli (or E. coli for short), are used as factories for the production of therapeutics and other compounds of commercial or clinical value; much in the same way that yeast can be used to make alcohol. All organisms contain blocks of information called genes, which are passed from one generation to the next. The process by which the information in these genes is manifested in the characteristics of living organisms is called gene expression. The information in a gene is not read directly; instead, a copy called the messenger is made with a limited 'shelf life'. The shelf-life of the messenger determines how many times it can be read and, in turn, the characteristics of an organism. The stability of messengers in E. coli is the subject area of this application. More specifically, we wish to characterise a newly identified mode of recognising messengers that evidence from a number of sources indicates is pivotal to the process of initiating their degradation. We also plan to determine whether this mode of recognition can explain why messengers are highly vulnerable to degradation when they are not being read. This happens, for example, upon the binding of messengers by small RNAs, which represent an emerging class of regulator that is associated with bacterial survival and pathogenicity. In the longer term, our research may also impact the search for new antimicrobials, which is timely given the alarming rate at which resistance to antibiotics is emerging in bacterial populations. The function of E. coli RNase E is essential, so disabling its activity could be lethal to the many pathogens that contain this enzyme. Knocking out RNase E activity after E. coli has grown (in the form of a bio-factory) may also lead to the increased production of compounds of commercial and clinical value, by increasing the number of times the corresponding messengers can be read. This proposal is also relevant to synthetic biology, 'a new and growing science that focuses on re-designing and re-building natural biological systems synthetically from the ground up'. In the 32nd annual Richard Dimbleby lecture, Dr. J. Craig Venter (who is probably best known for heading the private enterprise that sequenced the human genome) outlined how bacteria could be engineered by 'human intelligence' to create renewable energy and combat climate change. This is not science fiction. Modified bacteria are already being used by DuPont to convert sugar into a new polymer that can be used to produce stain-resistant carpets and clothing. Other companies including BP are trying to adapt bacteria to make the next generation of biofuels. Genes with related functions (e.g. those that part of the same pathway or complex) can be expressed as part of the same messenger in bacteria. This ensures that all the components for a particular process are made at the same time. The ultimate goal for synthetic biologists is to be able to design systems from 'scratch' using rules learnt from nature. In these systems, the messengers will need to have a sufficient 'shelf-life' to be able to confer attributes that are desired, while at the same time not being too stable such that the information is permanent. This proposal could provide information necessary for the rational engineering of messengers encoding multiple genes such that different components can be made in different amounts.

Recently, we have published findings that impact our view of mRNA degradation in E. coli and the many organisms that contain homologues of RNase E. For over a decade, this endonuclease has been synonymous with the description '5'-end dependent'. Indeed, it was thought that a 5'-monophosphate (5'P) was critical as an allosteric activator. However, we have shown that the N-terminal catalytic half of RNase E cleaves certain RNAs rapidly irrespective of the status of their 5' end. Moreover, the minimum substrate requirement for this mode of cleavage, which can be categorised as 'direct' entry, appears to be multiple single-stranded segments in a conformational context that allows their simultaneous interaction with RNase E. While previous work has hinted at a possible role for direct entry, the relative simplicity of these requirements suggests that it could represent a major means of initiating mRNA degradation. This mode of recognition also suggests an exquisitely simple surveillance mechanism by which the degradation of mRNAs that are translationally defective or repressed would be initiated at an accelerated rate. Importantly, our model is supported by a collaborator who has shown that 5' sensing is not required for the essential activity of RNase E, and by the finding that deletion of the pyrophosphatase that generates the 5'P group on nascent transcripts does not result in the stabilisation of the major proportion of E. coli mRNAs. We propose now to (i) characterise the molecular recognition (determine the 'code') that underlies direct entry including its activation by the C-terminal half of RNase E, which serves as a platform for degradosome assembly, (ii) establish experimentally the extent to which 5'P-independent cleavage impacts the degradation of the entire mRNA pool, and to (iii) investigate the interplay with translation with regard to non-nucleolytic inactivation by small RNAs, an emerging class of regulators linking to bacterial survival and virulence.

THOSE WHO WILL BENEFIT FROM THIS RESEARCH This proposal is likely to have high impact as it is based on an unprecedented finding related to the modus operandi of RNase E, the major initiator nuclease of mRNA degradation in E. coli. Thus, the completion of the proposed research should benefit those wishing to understand and manipulate the processes of gene regulation. As detailed in the previous section, it will benefit a number of collaborations and the research of a number of groups worldwide that study bacterial mRNA turnover. It should also have wider academic benefit in areas such as nucleic acid-protein interactions, computational modelling of cell-based systems and developmental biology. Beneficiaries may also include the commercial private sector that produces proteins using recombinant DNA technology and the wider public through improved health and wellbeing. HOW THEY WILL BENEFIT FROM THIS RESEARCH E. coli is used extensively as a 'factory' for the production of heterologous proteins, including those with commercial and clinical value. Many of the protein production systems use T7 RNA polymerase to direct transcription. However, it has been shown that protein production by this system can be limited by the rapid degradation of the mRNA. This system has been improved by using strains that lack the CTH of RNase E (e.g. BL21 Star (DE3) cells), but the production of protein can still be problematic. As detailed in this application, it may be more efficient to block the activity of the N-terminal catalytic half. Our research could inform the design of decoy RNAs or the selection of small molecules that will protect heterologous mRNA from RNase E attack. Thus, our research has potential economic benefit to the nation's wealth in the medium term (5-10 y). The study of mRNA degradation may also reveal potential targets for new antimicrobials. RNase E, which is essential, is being targeted by us and others. Thus, there may be benefits to public health and wellbeing in the longer term (>10 y). HOW THE PROPOSED RESEARCH PROJECT WILL BE MANAGED TO ENGAGE USERS AND BENEFICIARIES AND INCREASE THE LIKELIHOOD OF IMPACTS IP stemming from this proposal will be managed by the applicants with the assistance of Techtran Group Limited, a technology transfer company that provides services to the University of Leeds. It not only offers expertise in the identification of novel intellectual property with commercial potential, but seed capital to finance spin out companies and ongoing strategic and financial support to maximise the chances of success. Inhibitors of RNase E resulting from screens informed by, but not part of, this application (details withheld because of commercial sensitivity) can be assessed for clinical value through our Biomedical Health Research Centre, a joint venture by the University of Leeds and Leeds Teaching Hospitals NHS Trust. The remit of BHRC is ensure the translation of research from the laboratory to the clinic by bringing together basic scientists with not only clinicians but also with experts from economics, social and other health-relevant disciplines to ensure the full potential of basic research is realised. The applicants will also disseminate their findings, when appropriate, through publication in scientific journals and presentations at national and international meetings. Often the latter are attended by companies such as Invitrogen, Agilent and Qiagen, who may well be interest in advances that improve the production of recombinant protein, and PTC Therapeutics (New Jersey), a leader in the development of drugs that target post-transcriptional gene control . The principal applicant is currently organising such a meeting as part of the FASEB program. The findings will also be disseminated to the general public through newspaper articles, university open days and engagement with local schools or youth organisations. The principal applicant has participated in all of these activities