Polyamine sensing during antizyme mRNA programmed frameshifting

A key regulator of cellular polyamine levels from yeasts to mammals is the protein antizyme. The antizyme gene consists of two overlapping reading frames with ORF2 in the +1 frame relative to ORF1. A programmed +1 ribosomal frameshift occurs at the last codon of ORF1 and results in the production of full-length antizyme protein. The efficiency of frameshifting is proportional to the concentration of polyamines, thus creating an autoregulatory circuit for controlling polyamine levels. The mRNA recoding signals for frameshifting include an element 5' and a pseudoknot 3' of the shift site. The present work illustrates that the ORF1 stop codon and the 5' element are critical for polyamine sensing, whereas the 3' pseudoknot acts to stimulate frameshifting in a polyamine independent manner. We also demonstrate that polyamines are required to stimulate stop codon readthrough at the MuLV redefinition site required for normal expression of the GagPol precursor protein.     

Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.     

Antisense-induced ribosomal frameshifting

Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1–ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem–loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5′ of the shift site and to polyamine levels.     

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.     

Identification of an archaeal alpha-L-fucosidase encoded by an interrupted gene. Production of a functional enzyme by mutations mimicking programmed -1 frameshifting

The analysis of the complete genome of the thermoacidophilic Archaeon Sulfolobus solfataricus revealed two open reading frames (ORF), named SSO11867 and SSO3060, interrupted by a -1 frameshift and encoding for the N- and the C-terminal fragments, respectively, of an alpha-l-fucosidase. We report here that these ORFs are actively transcribed in vivo, and we confirm the presence of the -1 frameshift between them at the cDNA level, explaining why we could not find alpha-fucosidase activity in S. solfataricus extracts. Detailed analysis of the region of overlap between the two ORFs revealed the presence of the consensus sequence for a programmed -1 frameshifting. Two specific mutations, mimicking this regulative frameshifting event, allow the expression, in Escherichia coli, of a fully active thermophilic and thermostable alpha-l-fucosidase (EC ) with micromolar substrate specificity and showing transfucosylating activity. The analysis of the fucosylated products of this enzyme allows, for the first time, assigning a retaining reaction mechanism to family 29 of glycosyl hydrolases. The presence of an alpha-fucosidase putatively regulated by programmed -1 frameshifting is intriguing both with respect to the regulation of gene expression and, in post-genomic era, for the definition of gene function in Archaea.     

The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames

The L-A double-stranded RNA virus of Saccharomyces cerevisiae encodes its major coat protein (80 kDa) and a minor single-stranded RNA binding protein (180 kDa) that has immunological cross-reactivity with the major coat protein. The sequence of L-A cDNA clones revealed two open reading frames (ORF), ORF1 and ORF2. These two reading frames overlap by 130 base pairs and ORF2 is in the -1 reading frame with respect to ORF1. Although the major coat protein of the viral particles is encoded by ORF1, the 180-kDa protein is derived from the entire double-stranded RNA genome by fusing ORF1 and ORF2, probably by a -1 translational frameshift. Within the overlapping region is a sequence similar to that producing a -1 frameshift by "simultaneous slippage" in retroviruses. The coding sequence of ORF2 shows a pattern characteristic of viral RNA-dependent RNA polymerases of icosahedral (+)-strand RNA viruses. Thus, the 180-kDa protein is analogous to gag-pol fusion proteins.     

Structural requirements for efficient translational frameshifting in the synthesis of the putative viral RNA-dependent RNA polymerase of potato leafroll virus.

The putative RNA-dependent RNA polymerase of potato leafroll luteovirus (PLRV) is expressed by -1 ribosomal frameshifting in the region where the open reading frames (ORF) of proteins 2a and 2b overlap. The signal responsible for efficient frameshift is composed of the slippery site UUUAAAU followed by a sequence that has the potential to adopt two alternative folding patterns, either a structure involving a pseudoknot, or a simple stem-loop structure. To investigate the structure requirements for efficient frameshifting, mutants in the stem-loop or in the potential pseudoknot regions of a Polish isolate of PLRV (PLRV-P) have been analyzed. Mutations that are located in the second stem (S2) of the potential pseudoknot structure, but are located in unpaired regions of the alternative stem-loop structure, reduce frameshift efficiency. Deletion of the 3' end sequence of the alternative stem-loop structure does not reduce frameshift efficiency. Our results confirm that -1 frameshift in the overlap region depends on the slippery site and on the downstream positioned sequence, and propose that in PLRV-P a pseudoknot is required for efficient frameshifting. These results are in agreement with those recently published for the closely related beet western yellows luteovirus (BWYV).     

Triple decoding of hepatitis C virus RNA by programmed translational frameshifting

Ribosomes can be programmed to shift from one reading frame to another during translation. Hepatitis C virus (HCV) uses such a mechanism to produce F protein from the -2/+1 reading frame. We now report that the HCV frameshift signal can mediate the synthesis of the core protein of the zero frame, the F protein of the -2/+1 frame, and a 1.5-kDa protein of the -1/+2 frame. This triple decoding function does not require sequences flanking the frameshift signal and is apparently independent of membranes and the synthesis of the HCV polyprotein. Two consensus -1 frameshift sequences in the HCV type 1 frameshift signal facilitate ribosomal frameshifts into both overlapping reading frames. A sequence which is located immediately downstream of the frameshift signal and has the potential to form a double stem-loop structure can significantly enhance translational frameshifting in the presence of the peptidyl-transferase inhibitor puromycin. Based on these results, a model is proposed to explain the triple decoding activities of the HCV ribosomal frameshift signal.     

A retrotransposon-derived gene, PEG10, is a novel imprinted gene located on human chromosome 7q21

A novel paternally expressed imprinted gene, PEG10 (Paternally Expressed 10), was identified on human chromosome 7q21. PEG10 is located near the SGCE (Sarcoglycan epsilon) gene, whose mouse homologue was recently shown to be imprinted. Therefore, it is highly possible that a new imprinted gene cluster exists on human chromosome 7q21. Analysis of two predicted open reading frames (ORF1 and ORF2) revealed that ORF1 and ORF2 have homology to the gag and pol proteins of some vertebrate retrotransposons, respectively. These data suggest that PEG10 is derived from a retrotransposon that was previously integrated into the mammalian genome. PEG10 is likely to be essential for understanding how exogenous genes become imprinted.     

Evolutionary specialization of recoding: frameshifting in the expression of S. cerevisiae antizyme mRNA is via an atypical antizyme shift site but is still +1

An autoregulatory translational shift to the +1 frame is required for the expression of ornithine decarboxylase antizyme from fungi to mammals. In most eukaryotes, including all vertebrates and a majority of the studied fungi/yeast, the site on antizyme mRNA where the shift occurs is UCC-UGA. The mechanism of the frameshift on this sequence likely involves nearly universal aspects of the eukaryotic translational machinery. Nevertheless, a mammalian antizyme frameshift cassette yields predominantly -2 frameshift in Saccharomyces cerevisiae, instead of the +1 in mammals. The recently identified endogenous S. cerevisiae antizyme mRNA has an atypical shift site: UGC-GCG-UGA. It is shown here that endogenous S. cerevisiae antizyme frameshifting is +1 rather than -2. We discuss how antizyme frameshifting in budding yeasts exploits peculiarities of their tRNA balance, and relate this to prior studies on Ty frameshifting.     

An 'elaborated' pseudoknot is required for high frequency frameshifting during translation of HCV 229E polymerase mRNA.

The RNA polymerase gene (gene 1) of the human coronavirus 229E is approximately 20 kb in length and is located at the 5' end of the positive-strand genomic RNA. The coding sequence of gene 1 is divided into two large open reading frames, ORF1a and ORF1b, that overlap by 43 nucleotides. In the region of the ORF1a/ORF1b overlap, the genomic RNA displays two elements that are known to mediate (-1) ribosomal frameshifting. These are the slippery sequence, UUUAAAC, and a 3' pseudoknot structure. By introducing site-specific mutations into synthetic mRNAs, we have analysed the predicted structure of the HCV 229E pseudoknot and shown that besides the well-known stem structures, S1 and S2, a third stem structure, S3, is required for a high frequency of frameshifting. The requirement for an S3 stem is independent of the length of loop 2.     

Release factor 2 frameshifting sites in different bacteria

The mRNA encoding Escherichia coli polypeptide chain release factor 2 (RF2) has two partially overlapping reading frames. Synthesis of RF2 involves ribosomes shifting to the +1 reading frame at the end of the first open reading frame (ORF). Frameshifting serves an autoregulatory function. The RF2 gene sequences from the 86 additional bacterial species now available have been analyzed. Thirty percent of them have a single ORF and their expression does not require frameshifting. In the ~70% that utilize frameshifting, the sequence cassette responsible for frameshifting is highly conserved. In the E. coli RF2 gene, an internal Shine–Dalgarno (SD) sequence just before the shift site was shown earlier to be important for frameshifting. Mutagenic data presented here show that the spacer region between the SD sequence and the shift site influences frameshifting, and possible mechanisms are discussed. Internal translation initiation occurs at the shift site, but any functional role is obscure.     

Differential response to frameshift signals in eukaryotic and prokaryotic translational systems.

The genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a heptanucleotide slippery sequence and a downstream pseudoknot, is similar in appearance to those identified in retroviral RNAs. We have examined whether the proposed BWYV signal functions in frameshifting in three translational systems, i.c. in vitro in a reticulocyte lysate or a wheat germ extract and in vivo in E. coli. The efficiency of the signal in the eukaryotic system is low but significant, as it responds strongly to changes in either the slip sequence or the pseudoknot. In contrast, in E. coli there is hardly any response to the same changes. Replacing the slip sequence to the typical prokaryotic signal AAAAAAG yields more than 5% frameshift in E. coli. In this organism the frameshifting is highly sensitive to changes in the slip sequence but only slightly to disruption of the pseudoknot. The eukaryotic assay systems are barely sensitive to changes in either AAAAAAG or in the pseudoknot structure in this construct. We conclude that eukaryotic frameshift signals are not recognized by prokaryotes. On the other hand the typical prokaryotic slip sequence AAAAAAG does not lead to significant frameshifting in the eukaryote. In contrast to recent reports on the closely related potato leafroll virus (PLRV) we show that the frameshifting in BWYV is pseudoknot-dependent.     

Ribosomal frameshifting requires a pseudoknot in the Saccharomyces cerevisiae double-stranded RNA virus.

The large double-stranded RNA of the Saccharomyces cerevisiae (yeast) virus has two large overlapping open reading frames on the plus strand, one of which is translated via a -1 ribosomal frameshift. Sequences including the overlapping region, placed in novel contexts, can direct ribosomes to make a -1 frameshift in wheat germ extract, Escherichia coli and S. cerevisiae. This sequence includes a consensus slippery sequence, GGGUUUA, and has the potential to form a pseudoknot 3' to the putative frameshift site. Based on deletion analysis, a region of 71 nucleotides including the potential pseudoknot and the putative slippery sequence is sufficient for frameshifting. Site-directed mutagenesis demonstrates that the pseudoknot is essential for frameshifting.