Identification and characterisation of a developmentally regulated mammalian gene that utilises –1 programmed ribosomal frameshifting

Kazuhiro Shigemoto 2 Jane Brennan 2 Elizabeth Walls 2 Christine J. Watson 1 David Stott 0 Peter W. J. Rigby 0 1 Alastair D. Reith 0 1 2 0 Division of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research , The Ridgeway, Mill Hill, NW7 1AA, UK 1 CRC Eukaryotic Molecular Genetic Research Group, Department of Biochemistry, Imperial College of Science and Technology , London SW7 2AZ, UK 2 Ludwig Institute for Cancer Research , 91 Riding House Street, London W1P 8BT, UK Translational recoding of mRNA through a -1 ribosomal slippage mechanism has been observed in RNA viruses and retrotransposons of both eukaryotes and prokaryotes. Whilst this provides a potentially powerful mechanism of gene regulation, the utilization of -1 translational frameshifting in regulating mammalian gene expression has remained obscure. Here we report a mammalian gene, Edr, which provides the first example of -1 translational recoding in a eukaryotic cellular gene. In addition to bearing functional frameshift elements that mediate expression of distinct polypeptides, Edr bears both CCHC zinc-finger and putative aspartyl protease catalytic site retroviral-like motifs, indicative of a relic retroviral-like origin for Edr. These features, coupled with conservation of Edr as a single copy gene in mouse and man and striking spatio-temporal regulation of expression during embryogenesis, suggest that Edr plays a functionally important role in mammalian development. - In all species, triplet codon sequences on mRNA are accurately translated into polypeptides through recognition by aminoacyl tRNAs, such that the open reading frame (ORF) of the mRNA is maintained. In addition, non-standard decoding mechanisms have been identified in the form of programmed translational frameshifting, in which translating ribosomes are induced to slide 1 nt forward (+1 frameshifting) or backwards (1 frameshifting) at a specific recoding site on the mRNA. As a consequence, multiple ORFs can be utilised from a single mRNA so diversifying the polypeptide coding sequence information of gene transcription units. Programmed frameshifting has been found to occur in a variety of viral genomes (13), but is rare amongst prokaryotic and eukaryotic cellular genes. +1 programmed frameshifting has been observed in the prfB gene of Escherichia coli, which encodes peptide release factor 2 (RF2), and mammalian ornithine decarboxylase (ODC) antizymes (48). Interestingly, RF2 and antizyme genes use +1 frameshifting to modulate their own expression by negative feedback mechanisms. The concentration of RF2 products directly regulates the efficiency of such programmed frameshifting. ODC is the first and rate-limiting enzyme in the pathway of polyamine biosynthesis in mammalian cells. Antizymes not only inhibit the enzymatic activity of ODC but also mediate its degradation. The efficiency of frameshifting by antizyme genes that is required to synthesise the active form of antizyme is governed by the amount of polyamine present. These regulatory mechanisms of ODC are conserved from yeast to humans (9). Programmed 1 frameshifting is much more prevalent among viruses than other species. For example, retroviruses, coronaviruses, toroviruses, arteriviruses and paromyxoviruses of mammals all utilise programmed frameshifts in decoding compact genomes (1,2,10). Programmed 1 ribosomal frameshifting in viruses conforms to a single model in which an N- and C-terminal fusion protein is encoded by two distinct, overlapping ORFs. Two basic sequence elements are required to promote efficient levels of 1 frameshifting. The first is a slippery heptamer sequence, X XXY YYZ (the 0 frame is indicated by the spaces) that allows the simultaneous slippage of ribosome-bound A- and P-site tRNAs by one base in the 5 direction. The second is a RNA structural element downstream from the slippery sequence, consisting of either a pseudoknot structure or a simple stemloop, which induces a ribosomal pause over the slippery site and increases the probability of 5 ribosomal movement. In E.coli, the dnaX gene utilises 1 translational frameshifting as a regulatory mechanism for coordinated expression of and subunits of DNA polymerase III holoenzyme (1113). The dnaX frameshifting occurs on the slippery heptamer sequence, A AAA AAG, stimulated by two elements; an upstream hairpin and an upstream Shine Dalgarno interaction site. Amongst eukaryotes, a recent bioinformatics based approach searching for consensus 1 ribosomal frameshift signals, including a heptamer sequence X XXY YYZ and a putative RNA pseudoknot structure, identi fied a number of cellular genes from several species including human, mouse, rat, chick and yeast with the potential to utilise 1 translational frameshifting (14). In a number of cases, identified frameshift signals were conserved in homologous genes from separate species. For example, the activity of 1 frameshifting in Saccharomyces cerevisiae was demonstrated in two selected motifs from yeast RAS1 and human CCR5 mRNA. However, these genes contained frameshift signals positioned to cause premature stoppage of the reading frames (10). Thus, the significance and origin of 1 frameshifting in these examples remains obscure. Here we report a single copy mammalian gene, Edr (embryonal carcinoma differentiation regulated), which utilises a 1 ribosomal recoding mechanism to encode distinct polypeptides. Together with the presence of a CCHC zinc-binding motif and a putative aspartyl protease catalytic site, Edr is a new member of an emerging class of eukaryotic genes that bear relic motifs of retroviral genomes. The conservation of Edr in mouse and human, together with spatially and temporally regulated expression during embryogenesis suggest that Edr may play an important role in mammalian development. MATERIALS AND METHODS Isolation of Edr cDNA clones and northern blot analysis The 2.4 kb partial Edr cDNA clone pGR165 was identified as one of several clones isolated by differential screening of a PCC3 cDNA library, as described previously (15). Total RNA was prepared from PCC3 cells, mouse embryos and adult tissues. The probe DNA was prepared from a 2.4 kb Edr cDNA clone (pGR165) isolated from PCC3 cDNA library, and labelled with [-32P]dCTP. Mouse HPRT cDNA clone pHPT4 has been reported previously. Human -actin cDNA was purchased from Clontech. The cDNA insert of pGR165 was used to screen 4 l05 clones from an E12.5 CD1 mouse embryo cDNA library (ZapII). Phage DNA was screened with the radiolabelled probe for 12 h at 42C in 1 hybridization buffer (50 mM phosphate buffer pH 7.5, 5 SSC, 4 Denhardts reagent and 100 g/ml herring sperm DNA, and 50% [v/v] formaldehyde), and washed twice for 30 min at 60C in 0.2 SSC, 0.1% [v/v] SDS. Nested deletion derivatives of cDNA clones were obtained by unidirectional deletion using exonuclease III. Nucleotide sequence determination was performed by chain termination method using [-35S]dATP (Amersham (...truncated)