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KnotInFrame: prediction of -1 ribosomal frameshift events.

ABSTRACT: Programmed -1 ribosomal frameshift (-1 PRF) allows for alternative reading frames within one mRNA. First found in several viruses, it is now believed to exist in all kingdoms of life. Strong stimulators for -1 PRF are a heptameric slippery site and an RNA pseudoknot. Here, we present a new algorithm KnotInFrame, for the automatic detection of -1 PRF signals from genomic sequences. It finds the frameshifting stimulators by means of a specialized RNA-pseudoknot folding program, fast enough for genome-wide analyses. Evaluations on known -1 PRF signals demonstrate a high sensitivity.

SUBMITTER: Theis C

PROVIDER: S-EPMC2566878 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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KnotInFrame: prediction of -1 ribosomal frameshift events.

Theis Corinna C Reeder Jens J Giegerich Robert R

Nucleic acids research 20080927 18

Programmed -1 ribosomal frameshift (-1 PRF) allows for alternative reading frames within one mRNA. First found in several viruses, it is now believed to exist in all kingdoms of life. Strong stimulators for -1 PRF are a heptameric slippery site and an RNA pseudoknot. Here, we present a new algorithm KnotInFrame, for the automatic detection of -1 PRF signals from genomic sequences. It finds the frameshifting stimulators by means of a specialized RNA-pseudoknot folding program, fast enough for gen ...[more]

PMID: 18820303

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programmed ribosomal frameshift

Project description:programmed ribosomal frameshift Raw sequence reads

| PRJNA310019 | ENA

FSscan: a mechanism-based program to identify +1 ribosomal frameshift hotspots.

Project description:In +1 programmed ribosomal frameshifting (PRF), ribosomes skip one nucleotide toward the 3'-end during translation. Most of the genes known to demonstrate +1 PRF have been discovered by chance or by searching homologous genes. Here, a bioinformatic framework called FSscan is developed to perform a systematic search for potential +1 frameshift sites in the Escherichia coli genome. Based on a current state of the art understanding of the mechanism of +1 PRF, FSscan calculates scores for a 16-nt window along a gene sequence according to different effects of the stimulatory signals, and ribosome E-, P- and A-site interactions. FSscan successfully identified the +1 PRF site in prfB and predicted yehP, pepP, nuoE and cheA as +1 frameshift candidates in the E. coli genome. Empirical results demonstrated that potential +1 frameshift sequences identified promoted significant levels of +1 frameshifting in vivo. Mass spectrometry analysis confirmed the presence of the frameshifted proteins expressed from a yehP-egfp fusion construct. FSscan allows a genome-wide and systematic search for +1 frameshift sites in E. coli. The results have implications for bioinformatic identification of novel frameshift proteins, ribosomal frameshifting, coding sequence detection and the application of mass spectrometry on studying frameshift proteins.

| S-EPMC2790909 | biostudies-literature

Structural analysis of the -1 ribosomal frameshift elements in giardiavirus mRNA.

Project description:The RNA polymerase of giardiavirus (GLV) is synthesized as a fusion protein through a -1 ribosomal frameshift in a region where gag and pol open reading frames (ORFs) overlap. A heptamer, CCCUUUA, and a potential pseudoknot found in the overlap were predicted to be required for the frameshift. A 68-nucleotide (nt) cDNA fragment containing these elements was inserted between the GLV 5' 631-nt cDNA and the out-of-frame luciferase gene that required a -1 frameshift within the 68-nt fragment for expression. Giardia lamblia trophozoites transfected with the transcript of this construct showed a frameshift frequency at 1.7%, coinciding with the polymerase-to-capsid protein ratio in GLV. The heptamer is required for the frameshift but can be replaced with other sequences of the same motif. Mutations placing stop codons in the 0 or -1 frame, located directly before or after the heptamer, implicated the latter as the site for the -1 frameshift. Shortening or destroying the putative stem decreased the frameshift efficiency threefold; the efficiency was fully recovered by mutations to restore the stem. Deleting 18 nt from the 3' end of the 68-nt fragment, which formed the second stem in the putative pseudoknot, had no effect on the frequency of the frameshift. Chemical probing of the RNA secondary structure in the frameshift region showed that bases resistant to chemical modification were clustered in the putative stem structures, thus confirming the presence of the postulated stem-loop, while all the bases in the loop were chemically modified, thus ruling out their capability of forming a pseudoknot. These results confirmed the conclusion based on data from the mutation study that there is but a simple stem-loop downstream from the heptamer. Together, they constitute the structural elements for a -1 ribosomal frameshift in the GLV transcript.

| S-EPMC114643 | biostudies-literature

Characterization of the frameshift stimulatory signal controlling a programmed -1 ribosomal frameshift in the human immunodeficiency virus type 1.

Project description:Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshifting when ribosomes translate the unspliced viral messenger RNA. This frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates frameshifting. This motif is commonly assumed to be a simple stem-loop for HIV-1. In this study, we show that the frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem-loop corresponds to the classic stem-loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem-loop with a segment downstream of this stem-loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol frameshift region of HIV-1. The involvement of the novel lower stem in frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a -1 frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced frameshifting, whereas compensatory changes that allow re-formation of this stem restored the frameshift efficiency near wild-type level. The two-stem structure that we propose for the frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.

| S-EPMC137970 | biostudies-literature

rRNA-mRNA base pairing stimulates a programmed -1 ribosomal frameshift.

Project description:Base pairing between the 3' end of 16S rRNA and mRNA is shown to be important for the programmed -1 frameshifting utilized in decoding the Escherichia coli dnaX gene. This pairing is the same as the Shine-Dalgarno pairing used by prokaryotic ribosomes in selection of translation initiators, but for frameshifting the interaction occurs within elongating ribosomes. For dnaX -1 frameshifting, the 3' base of the Shine-Dalgarno sequence is 10 nucleotides 5' of the shift site. Previously, Shine-Dalgarno rRNA-mRNA pairing was shown to stimulate the +1 frameshifting necessary for decoding the release factor 2 gene. However, in the release factor 2 gene, the Shine-Dalgarno sequence is located 3 nucleotides 5' of the shift site. When the Shine-Dalgarno sequence is moved to the same position relative to the dnaX shift site, it is inhibitory rather than stimulatory. Shine-Dalgarno interactions by elongating ribosomes are likely to be used in stimulating -1 frameshifting in the decoding of a variety of genes.

| S-EPMC197052 | biostudies-other

Selection of peptides interfering with a ribosomal frameshift in the human immunodeficiency virus type 1.

Project description:The human immunodeficiency virus of type 1 (HIV-1) uses a programmed -1 ribosomal frameshift to produce the precursor of its enzymes, and changes in frameshift efficiency reduce replicative fitness of the virus. We used a fluorescent two-reporter system to screen for peptides that reduce HIV-1 frameshift in bacteria, knowing that the frameshift can be reproduced in Escherichia coli. Expression of one reporter, the green fluorescent protein (GFP), requires the HIV-1 frameshift, whereas the second reporter, the red fluorescent protein (RFP), is used to assess normal translation. A peptide library biased for RNA binding was inserted into the sequence of the protein thioredoxin and expressed in reporter-containing bacteria, which were then screened by fluorescence-activated cell sorting (FACS). We identified peptide sequences that reduce frameshift efficiency by over 50% without altering normal translation. The identified sequences are also active against different frameshift stimulatory signals, suggesting that they bind a target important for frameshifting in general, probably the ribosome. Successful transfer of active sequences to a different scaffold in a eukaryotic test system demonstrates that the anti-frameshift activity of the peptides is neither due to scaffold-dependent conformation nor effects of the scaffold protein itself on frameshifting. The method we describe identifies peptides that will provide useful tools to further study the mechanism of frameshift and may permit the development of lead compounds of therapeutic interest.

| S-EPMC2327360 | biostudies-literature

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

Project description: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.

| S-EPMC1065257 | biostudies-literature

Identification of functional, endogenous programmed -1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae.

Project description:In viruses, programmed -1 ribosomal frameshifting (-1 PRF) signals direct the translation of alternative proteins from a single mRNA. Given that many basic regulatory mechanisms were first discovered in viral systems, the current study endeavored to: (i) identify -1 PRF signals in genomic databases, (ii) apply the protocol to the yeast genome and (iii) test selected candidates at the bench. Computational analyses revealed the presence of 10 340 consensus -1 PRF signals in the yeast genome. Of the 6353 yeast ORFs, 1275 contain at least one strong and statistically significant -1 PRF signal. Eight out of nine selected sequences promoted efficient levels of PRF in vivo. These findings provide a robust platform for high throughput computational and laboratory studies and demonstrate that functional -1 PRF signals are widespread in the genome of Saccharomyces cerevisiae. The data generated by this study have been deposited into a publicly available database called the PRFdb. The presence of stable mRNA pseudoknot structures in these -1 PRF signals, and the observation that the predicted outcomes of nearly all of these genomic frameshift signals would direct ribosomes to premature termination codons, suggest two possible mRNA destabilization pathways through which -1 PRF signals could post-transcriptionally regulate mRNA abundance.

| S-EPMC1802563 | biostudies-literature

Structural and functional conservation of the programmed -1 ribosomal frameshift signal of SARS-CoV-2.

Project description:17 years after the SARS-CoV epidemic, the world is facing the COVID-19 pandemic. COVID-19 is caused by a coronavirus named SARS-CoV-2. Given the most optimistic projections estimating that it will take over a year to develop a vaccine, the best short-term strategy may lie in identifying virus-specific targets for small molecule interventions. All coronaviruses utilize a molecular mechanism called -1 PRF to control the relative expression of their proteins. Prior analyses of SARS-CoV revealed that it employs a structurally unique three-stemmed mRNA pseudoknot to stimulate high rates of -1 PRF, and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity negatively impacts virus replication, suggesting that this molecular mechanism may be therapeutically targeted. Here we present a comparative analysis of the original SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar rates of -1 PRF and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablated -1 PRF activity. The upstream attenuator hairpin activity has also been functionally retained. Small-angle x-ray scattering indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 had the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may provide promising lead compounds to counter the current pandemic.

| S-EPMC7310627 | biostudies-literature

Conservation of the Escherichia coli dnaX programmed ribosomal frameshift signal in Salmonella typhimurium.

Project description:Escherichia coli DNA polymerase III subunits tau and gamma are produced from one gene, dnaX, by a programmed ribosomal frameshift which generates the C terminal of gamma within the tau reading frame. To help evaluate the role of the dispensable gamma, the distribution of tau and gamma homologs in several other species and the sequence of the Salmonella typhimurium dnaX were determined. All four enterobacteria tested produce tau and gamma homologs. S. typhimurium dnaX is 83% identical to E. coli dnaX, but all four components of the frameshift signal are 100% conserved.

| S-EPMC179275 | biostudies-other

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