Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting

Abstract

The ribosomal frameshifting signal of the mouse embryonal carcinoma differentiation regulated (Edr) gene represents the sole documented example of programmed -1 frameshifting in mammalian cellular genes [Shigemoto,K., Brennan,J., Walls,E,. Watson,C.J., Stott,D., Rigby,P.W. and Reith,A.D. (2001), Nucleic Acids Res., 29, 4079-4088]. Here, we have employed site-directed mutagenesis and RNA structure probing to characterize the Edr signal. We began by confirming the functionality and magnitude of the signal and the role of a GGGAAAC motif as the slippery sequence. Subsequently, we derived a model of the Edr stimulatory RNA and assessed its similarity to those stimulatory RNAs found at viral frameshift sites. We found that the structure is an RNA pseudoknot possessing features typical of retroviral frameshifter pseudoknots. From these experiments, we conclude that the Edr signal and by inference, the human orthologue PEG10, do not represent a novel 'cellular class' of programmed -1 ribosomal frameshift signal, but rather are similar to viral examples, albeit with some interesting features. The similarity to viral frameshift signals may complicate the design of antiviral therapies that target the frameshift process.

Figure 1

Construction of plasmid pKT0/Edr. A 1230 bp DNA fragment (631–1861) encompassing the Edr frameshift region was amplified by PCR from plasmid pSP64T/Edr (20) and cloned into NcoI/HindIII digested plasmid pKT0 (28). The 5′ and 3′ portions of the cloned Edr segment are shown in lower case, numbered according to the mRNA sequence (A of natural AUG start site is base 452; accession no. AJ006464). The AUG for the expression of Edr sequences in pKT0/Edr is derived from the vector (upper case, underlined). In ribosomal frameshifting assays, capped mRNAs were prepared by SP6 transcription of NdeI-linearized templates (unless otherwise stated). The predicted size of the non-frameshifted (stop) and frameshifted (fs) products generated from the translation of this mRNA in RRL is 29 and 32 kDa, respectively. For structural analysis of the Edr stimulatory RNA, a T3 promoter was introduced into the Edr sequence (at position 1357) to generate plasmid pKT0/Edr/T3. Linearization of this plasmid with NdeI and subsequent transcription with T3 RNA polymerase yields a transcript of 152 nt.

Figure 2

Confirming functionality of the Edr frameshift signal. pKT0/Edr was linearized with NdeI, AvrII, HindIII or BamHI, transcribed with T7 RNA polymerase and transcripts translated in RRL, either undiluted (1; final concentration ∼50 μg/ml) or diluted 1/3 (1/3; about 15 μg/ml). Products were labelled with [³⁵S]methionine, separated on a 15% SDS/polyacrylamide gel and detected by autoradiography. The non-frameshifted (stop) and frameshifted (FS) species are marked with arrows. M represents ¹⁴C protein markers (Amersham Pharmacia Biotech). C is a control translation of an mRNA derived from EcoRI-linearized plasmid p2luc/MMTV gag/pro (53). The first G of the putative Edr slippery sequence (GGGAAAC) is at position 1390 (in the mRNA sequence). The sites of cleavage of the four restriction endonucleases used above are 1501 (NdeI), 1764 (AvrII), 2028 (HindIII) and 2082 (BamH1) and are predicted to specify frameshift products of 33, 43, 52 and 54 kDa, respectively. The non-frameshifted product is predicted to be 29 kDa in each case. The frameshift efficiencies measured for each mRNA are shown (Fe%).

Figure 3

Deletion analysis of the Edr frameshift signal. (A) Three independent in-frame deletions were created in pKT0/Edr, Δ90, Δ63 and Δ24, to investigate the requirement for sequence information downstream of the putative Edr slippery sequence GGGAAAC (boxed). Each deletion (of 90, 63 or 24 nt) was to a common 3′ site (immediately downstream of the NdeI site, which was removed), leaving varying lengths of 5′ sequence. The nucleotides implicated as forming the stimulatory RNA in a previous study (20) are in bold. (B) The three deletion mutants were digested with HindIII, transcribed with SP6 RNA polymerase and transcripts translated in RRL, either undiluted (1; final concentration ∼50 μg/ml) or diluted 1/3 (1/3; about 15 μg/ml). Products were labelled with [³⁵S]methionine, separated on a 15% SDS/polyacrylamide gel and detected by autoradiography. The non-frameshifted (stop) and frameshifted (FS) species are marked with arrows. M represents ¹⁴C protein markers (Amersham Pharmacia Biotech). The species indicated with an asterisk is discussed in the text.

Figure 4

Proposed foldings of the Edr stimulatory RNA. (A) The stem–loop and pseudoknot models of Shigemoto et al. (20). (B) New pseudoknot model based on the results of the deletion analysis of Figure 3. In the new model, stem 1 is considered as two helices, 1a and 1b, separated by a 3 nt bulge (–ACA–) in the second arm.

Figure 5

Analysis of the Edr frameshift signal by site-directed mutagenesis. (A) A series of mutations were introduced into the Edr frameshift region to modify the proposed slippery sequence (GGGAAAC, in bold) or pseudoknot. (B). Wild-type pKT0/Edr or mutant derivatives were digested with HindIII, transcribed with SP6 RNA polymerase and transcripts translated in RRL at a concentration of ∼50 μg/ml. Products were labelled with [³⁵S]methionine, separated on 15% SDS/polyacrylamide gels and detected by autoradiography. The non-frameshifted (stop) and frameshifted (FS) species are marked with arrows. M represents ¹⁴C protein markers (Amersham Pharmacia Biotech). (C) Summary of the mutations made and the resulting frameshift efficiencies. In constructs pKT0/m5, m8 and m11, both arms of the relevant stem region were mutated such that the stems should reform (double/revertant).

Figure 6

Structure probing of the Edr frameshift signal. RNA derived by transcription of pKT0/Edr/T3/NdeI with T3 RNA polymerase was 5′ end-labelled with [γ-³³P]ATP and subjected to limited RNase or chemical cleavage using structure-specific probes. Sites of cleavage were identified by comparison with a ladder of bands created by limited alkaline hydrolysis of the RNA (OH⁻) and the position of known RNase U2 and T1 cuts, determined empirically. Products were analysed on a 6% acrylamide/7 M urea gel (A) or a 10% gel containing formamide (20% v/v) (B). Data were also collected from 6 to 15% gels (gels not shown). Enzymatic structure probing was with RNases CL3, T1, U2 and CV1. Uniquely cleaved nucleotides were identified by their absence in untreated control lanes (0). The number of units of enzyme added to each reaction is indicated, except in (B), the U2, T1 and CV1 reactions contained 0.1 U. Chemical structure probing was with imidazole (2 h, I) or lead acetate (Pb²⁺; mM concentration in reaction). The water lane (W) represents RNA, which was dissolved in water, incubated for 2 h and processed in parallel to the imidazole-treated sample. R represents an aliquot of the purified RNA loaded directly onto the gel without incubation in a reaction buffer.

Figure 7

Summary of the Edr probing results. (A) The sensitivity of bases in the Edr frameshift region to the various probes is shown. The size of the symbols is approximately proportional to the intensity of cleavage at that site. Bases in red indicate sequence differences present in the human orthologue, PEG10. (B) An alternative folding possibility for the slippery sequence/spacer/stem 1a region.