Non-canonical gene expression: Investigating a novel stimulator and a novel function for ribosomal frameshifting

Lead Research Organisation: University of Cambridge
Department Name: Pathology

Abstract

The central 'dogma' of molecular biology, articulated by Francis Crick in 1958, describes the transfer of information between the three major classes of information-carrying biopolymers: genetic information passes from one generation to the next via the replication of DNA and, within an organism, genes encoded within the DNA are transcribed into 'messenger' RNAs which are translated into proteins. Simple copying of DNA to DNA or DNA to RNA is mediated by molecular 'machines' known as polymerases. The far more complex process of translating proteins from messenger RNAs is mediated by a complex molecular machine known as the ribosome. Ribosomes are an essential component of all living organisms. Indeed the presence of ribosomes could be taken as a definition of life as we know it: even the simplest bacteria make their own ribosomes; in contrast, even the most complex viruses 'hijack' the ribosomes of their hosts.

DNA and RNA molecules comprise long strings of fours types of nucleotides which, for convenience, are denoted by the letters A, C, G and T (for DNA) and A, C, G and U (for RNA). The entire human genomes comprises ~3 billion nucleotides, within which are the 'instructions' to build ~25000 different proteins. Proteins comprise strings of amino acids, of which there are 20 standard types. To produce a protein from a messenger RNA, a ribosome reads consecutive groups of three nucleotides and translates the triplet into an amino acid, according to the 'genetic code'. However, in a proportion of genes in probably all organisms, specific motifs within messenger RNAs can stimulate a portion of ribosomes to deviate from standard translation. One type of exception is known as '-1 ribosomal frameshifting'. Here, at a specific site within a messenger RNA, a proportion of ribosomes deviate from reading consecutive triplets of nucleotides by 'slipping backward' by a single nucleotide. These ribosomes then continue to translate a series of triplets that are offset -1 nucleotide relative to other ribosomes that do not slip. Thus one messenger RNA can encode two completely different proteins. Ribosomal frameshifting is used by many viruses, such as HIV, SARS, West Nile, Japanese encephalitis and Porcine reproductive and respiratory syndrome viruses, and many more. Such viruses have very small genomes (~10000 to 25000 nucleotides) and ribosomal frameshifting plays a central role in allowing them to pack as much genetic information as possible into the available space. As such, ribosomal frameshifting plays a crucial role in the biology and virulence of many viruses.

Our research concerns a new case of ribosomal frameshifting that we recently discovered in a group of viruses known as the cardioviruses. Although not harbingers of dreadful doom, these viruses have been used extensively for medical and fundamental biological research (including to provide a model for multiple sclerosis). Thus, characterizing this previously undetected feature in these viruses will provide data that will likely aid reinterpretation of previous studies and allow for clearer interpretation of future results. However the main focus of our proposed research concerns the broader implications of understanding this particular case of frameshifting. This is because our previous research indicates that ribosomal frameshifting in the cardioviruses involves some fundamentally new mechanisms and very likely has some fundamentally new functional aspects. Investigating and characterizing these new features should shed new light on (a) mechanisms by which the ribosome may be induced to deviate from standard triplet decoding of messenger RNA to protein, (b) potential new aspects and functions of ribosomal frameshifting in other viruses including some of great public health import (HIV, SARS virus, West Nile, etc), and (c) the currently overall poorly understood, but clearly important, role of frameshifting in human genes.

Technical Summary

The translation of proteins from mRNAs is a central process in all cells. Characterizing exceptions to the rule of standard decoding plays an important role in understanding the mechanics of translation. One such exception, programmed -1 ribosomal frameshifting (-1 PRF), is used by many viruses to optimize the coding potential of compact genomes and to control gene expression. The degree to which PRF is functionally utilized by cellular genes remains uncertain, though recent work suggests that it may be used on a broad scale for fine-tuning mRNA turn-over. Thus, understanding the stimulatory mechanisms and biological functions of PRF is important for both virology and human biology.

Recently, we discovered a new and unusual case of -1 PRF in encephalomyocarditis virus (EMCV) and probably also in the related viruses, Theiler's murine encephalomyelitis (TMEV) and Saffold. In EMCV, PRF results in the translation of a novel 15 kDa transframe protein, 2B*, knock out of which leads to a small plaque phenotype. Unusually, PRF in EMCV is dependent on virus-infection, thus potentially providing the virus with a novel regulatory mechanism. Interestingly, TMEV maintains the frameshift site but lacks a long frameshift ORF, suggesting that here PRF may serve purely as a regulatory mechanism and/or as a ribosome sink.

The goals of this research are to characterize the cis and/or trans elements that stimulate PRF in EMCV and in TMEV, and to investigate the potential regulatory role or other functional significance of PRF in TMEV. Deciphering the apparently novel mechanism and potential regulatory role of PRF in these viruses has broad implications for (a) understanding the capacity of the ribosome to circumvent standard triplet decoding, (b) potential cellular cases of PRF, including regulated PRF, and (c) identifying potentially unappreciated roles of PRF in the molecular biology of other viruses. We will also investigate the function of the EMCV 2B* protein.

Planned Impact

This is a 'basic-research' project that will advance fundamental understanding of several complex biological processes and lay the groundwork for future advances in the following areas.

(1) Medicine, especially with respect to human TMEV-like viruses: Saffold cardiovirus is a very common human virus of early childhood, that nonetheless has only recently been characterized and whose clinical significance remains uncertain though it may be involved in enteric and/or respiratory disease (Zoll et al, 2009, PLoS Pathog, e1000416). The possibility that Saffold virus may be linked to human multiple sclerosis (in analogy to mouse-infecting TMEV) remains to be explored. Vilyuisk cardiovirus has been implicated in a severe neurological disease affecting people in the Vilyui River Valley in Russia. Thus cardioviruses are known human viruses of potential significance to health and further research into all aspects of their biology is relevant to health research, with consequent benefits for the general public.

(2) Human genome annotation and genetic diseases: Characterization of the nascent peptide, 3' RNA and/or trans-acting stimulators of cardiovirus frameshifting may lead to better prediction and a better understanding of short internal ORFs in cellular mRNAs that are functionally utilized via ribosomal frameshifting, with consequent medical implications for human genes that utilize such mechanisms, and potential treatments for genetic diseases.

(3) Virology and antiviral drugs: The research may lead to a deeper understanding of the mechanisms and roles of frameshifting in other viruses, including important human and livestock pathogens such as HIV, SARS virus, West Nile virus and Porcine respiratory and reproductive syndrome virus. This information could potentially lead to new antiviral drugs.

(4) Biotechnological tools: Non-canonical translation mechanisms have been and continue to be a source of valuable and broadly-applicable tools for molecular biological research (e.g. the EMCV IRES), with consequent economic benefits. Although such a use for the cardiovirus frameshift cassette appears relatively unlikely at the moment, it is a possibility that will be kept in mind as the research progresses.

Thus the potential beneficiaries are medical researchers in the public and private sector and through them the general public, besides the pharmaceutical and biotech industries.

The post doctoral research associate funded by this grant would acquire new expertise in molecular biological and virological research which would be widely applicable in the UK biotech industry. In addition, we would expect to have a number of short- and long-term students pass through the lab in the same time period and acquire skills of broad relevance to the UK's economy and well-being.

Publications

10 25 50
 
Description Many viruses utilize programmed -1 ribosomal frameshifting (-1 PRF) to access overlapping ORFs or to control gene expression. In nearly all studied cases, -1 PRF occurs on a slippery X_XXY_YYZ heptanucleotide sequence and is stimulated by 3'-adjacent intra-mRNA structure (either a stem-loop or a pseudoknot). Despite much effort searching for them, trans-acting stimulatory factors appear not to be required. In stark contrast, we previously showed that frameshifting on the cardiovirus frameshift sequence (e.g. fused into a reporter construct) critically depends on virus infection, suggesting the presence of a virus or virus-stimulated host trans-acting factor.

In Finch et al (2015, J Virol, PMID 26063423) we demonstrated that frameshifting occurs in TMEV (one of the grant objectives, as it had previously only been shown in EMCV). We measured, for the first time, the frameshifting efficiency in the context of the virus genome (as opposed to in reporter constructs) and found it to be an extraordinary 80% - the highest frameshifting efficiency in any virus, or in any mammalian system. We also showed that frameshifting depends on a stem-loop structure close to the frameshift site, but too far 3' to act in the normal way (it would be outside of the ribosome when the ribosome is positioned on the shift site). In Ling et al (2017, J Gen Virol, PMID 28786807) we performed some similar analyses in EMCV.

In Napthine et al (2017, Nat Commun, PMID 28593994) we completed the rest of the grant objectives and more besides. We used ribosome profiling to measure the frameshifting efficiency over a timecourse and found, uniquely, that it was not constant as in other viruses but increased from 0% at early timepoints to 70% at late timepoints. Using RiboTrap, we discovered that one of the virus proteins - 2A - binds to the stem-loop. This result was confirmed by EMSA and shown to be critically dependent on a positively charged tract in 2A and a CCC nucleotide sequence in the stem-loop. Using in vitro translations and virus mutants we showed that this RNA:protein interaction is necessary and sufficient for efficient ribosomal frameshifting in EMCV. This now provides the explanation for why frameshifting increases from 0% at early timepoints (little 2A in the cell) to 70% at late timepoints (high concentration of 2A in the cell). This nicely provides the virus with a way to maximize production of replication proteins at early timepoints but downregulate replication protein synthesis at late timepoints while still maintaining high levels of structural protein synthesis. There is potential relevance to other viruses that also utilize "single-polyprotein" gene expression strategies such as other picornaviruses and flaviviruses and we are investigating these posibilities. We are also investigating whether analogous examples of protein-regulated ribosomal frameshifting might also occur in some human genes.

Essentially all of the original grant objectives have been completed and published, though there still some additional unpublished results (manuscript in preparation). Through collaboration, this work is now progressing to biophysical and structural studies to further understand this novel mechanism of regulating protein synthesis, with potential biotechnological applications in the future.
Exploitation Route Following presentation at a conference, two European groups suggested to collaborate on further follow-up work using specialist techniques (now in progress); and a UK colleague obtained a 5-year Wellcome Trust Investigator Award for biophysical and structural follow-up studies (in progress, with some exciting recent results). We are also investigating certain biotechnological applications.
Sectors Pharmaceuticals and Medical Biotechnology,Other

URL http://www.firthlab.path.cam.ac.uk/index.html
 
Description The translation of proteins from mRNAs is a central process in all cells. Characterizing exceptions to the rule of standard decoding plays an important role in understanding the mechanics of protein synthesis. One such exception, programmed -1 ribosomal frameshifting (-1 PRF), is utilized in many virus genes (besides some cellular genes) to access overlapping reading frames and to control gene expression. Several years ago, we discovered a new and unusual case of -1 PRF in the cardiovirus encephalomyocarditis virus (EMCV). Unusually, PRF in EMCV is dependent on virus-infection, thus indicating a potential trans-acting factor, and potentially providing the virus with a novel regulatory mechanism. Interestingly, the related cardiovirus, Theiler's murine encephalomyelitis virus (TMEV), maintains the -1 PRF site but has only a very short frameshift ORF, suggesting that here PRF may serve purely as a regulatory mechanism. The main aims of the grant were to characterize the PRF mechanism in EMCV and in TMEV and investigate a potential regulatory role (giving new insight into the mechanics of protein synthesis and ribosomal frameshifting) and also assess whether the novel cardiovirus -1 PRF elements could be used as a biotechnological tool. During the course of the grant it became clear that the cardiovirus -1 PRF mechanism was very unusual indeed - being stimulated by a trans-acting viral protein - and also highly efficient (up to 80% of ribosomes make a -1 frameshift). A couple of potential biotechnological applications (for molecular biological research tools) are currently under investigation. Related to this and other projects in our lab, we have developed (and continue to expand) an online resource of virus gene expression data (http://www.firthlab.path.cam.ac.uk/vindex.html) which is linked-to from ViralZone and NCBI, and of use to the wider virology community.
First Year Of Impact 2014
Sector Other
Impact Types Economic

 
Description European Research Council Consolidators Grant
Amount € 1,780,000 (EUR)
Funding ID 646891 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 09/2015 
End 08/2020
 
Description Wellcome Trust Senior Research Fellowship
Amount £1,131,431 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2020
 
Title ViRAD database 
Description The Virus Re-Annotation Database (ViRAD) covers all RNA viruses and contains in-house reannotation of the coding regions based on homology, comparative genomics, expert review, and our own experimental analyses. The database also includes results of applying our in-house developed comparative genomic software to all RNA virus genomes. It is useful to the community for the identification of novel features in RNA virus genomes, which can be used to guide experimental analyses. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact NA 
URL http://www.firthlab.path.cam.ac.uk/virad.html
 
Description Extended studies of cardiovirus gene expression 
Organisation University College Cork
Country Ireland 
Sector Academic/University 
PI Contribution Sharing of research results.
Collaborator Contribution Reagents, expertise.
Impact Publications, conference presentations, grant funding.
Start Year 2010
 
Description Further analysis of TMEV mutants 
Organisation Catholic University of Louvain
Country Belgium 
Sector Academic/University 
PI Contribution Plasmids, research data.
Collaborator Contribution Reagents, facilities, expertise, research ideas.
Impact NA
Start Year 2013
 
Description Kinetic studies of cardiovirus frameshifting 
Organisation Max Planck Society
Department Max Planck Institute for Biophysical Chemistry Goettingen
Country Germany 
Sector Public 
PI Contribution Intellectual input.
Collaborator Contribution Intellectual input. Experimental work.
Impact No outcomes yet.
Start Year 2016
 
Description Single-molecule studies of cardiovirus frameshifting 
Organisation University Paris Sud
Country France 
Sector Academic/University 
PI Contribution Intellectual input.
Collaborator Contribution Intellectual input. Experimental work.
Impact No outcomes yet.
Start Year 2016
 
Description Structural studies of cardiovirus frameshifting mechanism 
Organisation Medical Research Council (MRC)
Department MRC Cognition and Brain Sciences Unit
Country United Kingdom 
Sector Public 
PI Contribution Intellectual input. Data processing. Staff training.
Collaborator Contribution Intellectual input. Experimental work. Equipment.
Impact Multidisciplinary: Bioinformatics + Biophysics + Molecular Biology. Grant funding: Investigator Award (funds 2 postdocs for 5 years) obtained by collaborator, half of which is to directly continue the work funded by the BBSRC grant.
Start Year 2016
 
Title RiboSeq Manual and Guide 
Description Software and user manual for analysing ribosome profiling data with a particularly emphasis on datasets that use ribosome profiling to study virus infection and virus gene expression 
Type Of Technology Software 
Year Produced 2017 
Open Source License? Yes  
Impact Have had several requests for the software and manual from researchers from a variety of fields (i.e. virus and otherwise). Recipients found the package very useful as it contains a wealth of information on our extensive experiences of using and analysing ribosome profiling data (e.g. artefacts to watch out for and how to interpret them). 
 
Title synplot2 comparative genomics software 
Description New comparative genomics software for mapping out functional RNA elements overlapping protein coding regions, focusing on, but not limited to, virus genomics. Source code and more recently (2014) a webserver interface made available to the community. 
Type Of Technology Software 
Year Produced 2012 
Open Source License? Yes  
Impact Has been used extensively in our lab for identifying novel functional elements encoded within the genomes of economically and medically important viruses such as influenza A virus, porcine reproductive and respiratory syndrome, West Nile virus and potyviruses. Many virus functional elements only exhibit a phenotype in animal models. By first identifying and characterizing features computationally, experimental follow-up can be targetted efficiently thus vastly reducing the need for any animal experiments. 
URL http://www.firthlab.path.cam.ac.uk/virad.html
 
Description American Society for Virology (ASV) conference, 21-25 Jul 2012, Madison, USA. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation paper presentation
Geographic Reach International
Primary Audience Other audiences
Results and Impact Conference talk by PI Andrew Firth. Changed people's views.

NA
Year(s) Of Engagement Activity 2012
URL http://conferencing.uwex.edu/conferences/asv2012/
 
Description BioProNet workshop on Protein Authenticity, London, 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Invited workshop talk by PI Andrew Firth, and followup discussion/interactions.
Year(s) Of Engagement Activity 2015
URL http://biopronetuk.org/
 
Description EMBO workshop on Recoding: Reprogramming genetic decoding, Killarney, Ireland, 13 - 18 May 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact PI Andrew Firth was a co-organizer of this workshop on Genetic Recoding. Much lively discussion between participants and forging of new networks and collaborations.

Hugely positive feedback from participants. Increased communication and knowledge sharing between practitioners of many different aspects of this growing field.
Year(s) Of Engagement Activity 2014
URL http://events.embo.org/14-recoding/
 
Description EMBO workshop on Recoding: Reprogramming genetic decoding, Killarney, Ireland, 13 - 18 May 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Conference talk by PI Andrew Firth. Lively discussion of our findings with many interested parties. Networking.

NA
Year(s) Of Engagement Activity 2014
URL http://events.embo.org/14-recoding/
 
Description European Virus Bioinformatics Centre 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited talk
Year(s) Of Engagement Activity 2017
URL http://evbc.uni-jena.de/
 
Description European Virus Bioinformatics Centre discussion session 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Co-chairing round-table discussion session on priorities in virus bioinformatics.
Year(s) Of Engagement Activity 2017
URL http://evbc.uni-jena.de/
 
Description Europic 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited talk
Year(s) Of Engagement Activity 2018
URL https://www.eiseverywhere.com/ehome/europic2018/442016/
 
Description Institut Pasteur - C3BI 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited seminar
Year(s) Of Engagement Activity 2017
 
Description Ribosome Profiling Workshop (London) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Presented and led a round table discussion session at a Biochemical Society workshop on Ribosome Profiling. This is a relatively new technique and the workshop was to share experiences between the different practitioners.
Year(s) Of Engagement Activity 2016
URL http://data.plantsci.cam.ac.uk/ribosome-footprinting/
 
Description Ribosome Structure and Function conference (Strasbourg) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Conference talk
Year(s) Of Engagement Activity 2016
URL http://events.embo.org/16-ribo/
 
Description Society for General Microbiology Annual Conference, 30 March - 2 April, 2015, Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Conference talk by PhD student Leanne Finch
Year(s) Of Engagement Activity 2015
URL http://www.microbiologysociety.org/conferences/annual-conferences/index.cfm/annual-conference-2015
 
Description Translation UK meeting, Aberdeen, 7-9 July, 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Conference talk by PI Andrew Firth
Year(s) Of Engagement Activity 2015
URL http://www.abdn.ac.uk/events/translational-uk-2015/
 
Description University College Cork 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited seminar
Year(s) Of Engagement Activity 2017
 
Description XIVth International Nidovirus Symposium 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited talk
Year(s) Of Engagement Activity 2017
URL https://www.vet.k-state.edu/Nido2017/