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

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.

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.

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.