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

Abstract

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.

Figure 1

Expression of Edr transcripts in PCC3 cells, adult mouse tissues and embryos. (A) Two micrograms of mRNA prepared from undifferentiated and differentiated PCC3 cells induced by RA for 4–10 days, were electrophoresed, blotted to nitrocellulose membrane and hybridised with [α-³²P]dCTP-labelled probes for Edr (pGR165) and HPRT. (B) Ten micrograms of total cellular RNA prepared from mouse embryos at 9.5 (E9.5) to 16.5 (E16.5) days gestation were electrophoresed, blotted to nitrocellulose membrane and hybridised with the same probes as above. (C) Ten micrograms of total cellular RNA prepared from each of adult heart (H), brain (B), spleen (S), lung (Ln), liver (L), skeletal muscle (SM), kidney (K) and testis (T) were electrophoresed, blotted to nitrocellulose membrane, and hybridised with probes for Edr (pGR165) and human β-actin cDNA. In the heart, 2 and 1.6kb isoforms of β-actin were observed, whereas a 1.6 kb isoform was detected in skeletal muscle.

Figure 2

Identification of retroviral-like motifs in Edr. (A) Amino acid sequence alignment of Edr and retroviral protease motifs. Consensus sequence of aspartyl protease active sites [LIVMFGAC]-[LIVMTADN]-[LIVFSA]-D-[ST]-G-[STAV]-[STAPDENQ]-X-[LIVMFSTNC]-[LIVMFGTA] are underlined. Conserved residues are shown in bold. HIV-1, human immunodeficiency virus type 1 (BH10 isolate); HIV-2, human immunodeficiency virus type 2 (ROD isolate); SIV, simian immunodeficiency virus (MAC isolate); MMLV, Moloney murine leukaemia virus; FELV, feline leukaemia virus; HTLV1, human T-cell leukaemia virus type I; HTLV2, human T-cell leukaemia virus type II; BLV, bovine leukaemia virus. (B) Diagrammatic representation of the predicted –1 ribosomal slippage heptamer and two models of RNA secondary structure downstream from the slippery sequence— a putative pseudoknot and a simple stem–loop structure. The slippage sequence, the potential pseudoknot and a simple stem–loop structures are shown in relation to predicted amino acid sequences of RF1 and RF2. The heptameric –1 slippage sequence is denoted in bold.

Figure 3

Edr utilises –1 ribosomal frameshifting to encode two polypeptides. (A) Plasmid templates for in vitro translation. pRF1, pRF2 and pRF1/RF2 expression plasmids were constructed by cloning Edr cDNA fragments into pSP65T as described in Materials and Methods. Plasmids pRF1/RF2-mt1 and pRF1/RF2-mt2 were derived from pRF1/RF2 by introduction of A→G and C→T nucleotide substitutions, respectively, within the putative slippage sequence. pRF1/RF2-mt3 was derived from pRF1/RF2-mt1 and contains an additional deletion of 2 nt predicted to contribute to the stable secondary structure of the pseudoknot structure. Shaded boxes denote the 5′- and 3′-untranslated regions within RF1 and RF2. The heptameric –1 slippage site is denoted in bold. (B) In vitro translation. Separate in vitro transcription and translation of plasmid templates from (A), was performed using rabbit reticulocyte lysate system in the presence of [³⁵S]methionine. Products were subjected to 10% SDS–PAGE prior to autoradiography.

Figure 4

Edr expression during musculo-skeletal development. Sagittal (A,C,D–F), or transverse (B) sections of E10.5 (A), E12.5 (B), E14.5 (C), E17.5 (D–F) mouse embryos were hybridised with antisense ³⁵S-radiolabelled RNA probes derived from the Edr cDNA clone pGR165, subjected to autoradiography for 48 h and counter-stained with toluidine blue. (A–C) 100× magnification. (D–F) 40× magnification. Some folding of tissue is apparent in (D). ASc, anterior sclerotome; Ca, calcaneus; Ch, chondrocytes; Cu, cuneiforme; D, dermatome; HCh, hypertrophied chondrocytes; IC, intercostal muscle; ID, intervertebral disc; M, myotome; Mt, cartilage primordium of metatarsale; Mwt, mesodermal web tissue; No, notochord; Pc, cutaneous muscle of thorax and trunk (panniculus carnosus); Po, periosteum; PSc, posterior sclerotome; Ra, radius; Sc, sclerotome; SM, fetal limb skeletal muscle; Ta, talus; Tm, Transversus thoracis muscle; VC, vertebral cartilage.

Figure 5

Edr is a conserved mammalian gene and maps to the proximal region of mouse chromosome 6. (A) Genomic Southern blot analysis: 10 µg genomic DNA prepared from human term placenta (hu) or M.musculus (mo) was digested with BamHI (B), XbaI (X) or EcoRI (E) restriction endonucleases, prior to electrophoresis and transfer to nitrocellulose. The filter was hybridised with ³²P-labelled Edr partial cDNA (395 bp EcoRI fragment encompassing nucleotides 1358–1753 in Edr cDNA) and washed to 0.1× SSC 0.1% SDS 65°C prior to autoradiography. (B) Comparison of the SDP of a BamHI RFLP at the Edr locus with others mapped in the BxD series of RI mice revealed linkage with D6Mit86 (0/26 recombinants and Met 4/26 recombinants). These linkage data map Edr to the proximal end of mouse chromosome 6.

Figure 6

Conservation of the –1 ribosomal frameshift signals and the aspartyl protease motif in human KIAA1051 gene. (A) Alignment of the nucleic acid sequences of Edr and KIAA1051 cDNAs. The heptameric –1 slippage sequence is denoted in bold. Conserved primary sequences for the potential pseudoknot and the simple stem–loop structures are shown in closed boxes, overlined (arrowed), respectively (KIAA1051 Genbank accession no. AB028974). (B) Comparison of the CCHC motif from Edr and KIAA1051. Conserved CCHC motifs are shown by asterisks. Basic residues outside CCHC motifs are marked with +. Identical residues are shaded. (C) Comparison of the putative aspartyl protease active sites from Edr and KIAA1051. Conserved residues for the aspartyl protease active sites are shown in bold. Identical residues are shaded.