Identification of programmed translational -1 frameshifting sites in the genome of Saccharomyces cerevisiae

Abstract

Frameshifting is a recoding event that allows the expression of two polypeptides from the same mRNA molecule. Most recoding events described so far are used by viruses and transposons to express their replicase protein. The very few number of cellular proteins known to be expressed by a -1 ribosomal frameshifting has been identified by chance. The goal of the present work was to set up a systematic strategy, based on complementary bioinformatics, molecular biology, and functional approaches, without a priori knowledge of the mechanism involved. Two independent methods were devised. The first looks for genomic regions in which two ORFs, each carrying a protein pattern, are in a frameshifted arrangement. The second uses Hidden Markov Models and likelihood in a two-step approach. When this strategy was applied to the Saccharomyces cerevisiae genome, 189 candidate regions were found, of which 58 were further functionally investigated. Twenty-eight of them expressed a full-length mRNA covering the two ORFs, and 11 showed a -1 frameshift efficiency varying from 5% to 13% (50-fold higher than background), some of which corresponds to genes with known functions. From other ascomycetes, four frameshifted ORFs are found fully conserved. Strikingly, most of the candidates do not display a classical viral-like frameshift signal and would have escaped a search based on current models of frameshifting. These results strongly suggest that -1 frameshifting might be more widely distributed than previously thought.

Figure 1.

Pipeline of frameshifting candidate identification strategy.

Figure 2.

Schematic representation of the genomic configurations compatible with -1 ribosomal frameshifting.

Figure 3.

Illustration of the HMM structures used for estimation and testing. (A) Estimating a suitable model for coding regions. The gene model estimated on all nonredundant ORFs of S. cerevisiae is fitting. (B) Estimating additional parameters before the filtering step, to test the possibility of a frameshifted coding region after the first stop (dashed arrow).

Figure 4.

Distribution of the probability of transition from the 0 to the -1 frame for the candidate regions compatible with a -1 frameshifting event. As evidenced in this distribution, a clear peak was observed at the 0.95 limit. This threshold value was thus chosen as a cutoff to choose the candidates to be ranked.

Figure 5.

A posteriori probabilities plot of the coding states of a “Bad” (fsORF 39; A) and “Good” (fsORF 28; B) candidate from the both subset. (Top) Symbolic representation of the reading frames (plain bars, stop codon; half bars, initiation codon). (Bottom) Probability of coding in each frame. Arrows indicate coding frames.

Figure 6.

RT-PCRs. Total RNA was extracted as described in the Methods and treated with DNase I. RT-PCR was carried out in two steps. First, reverse transcription was carried out using an oligo(dT) primer, allowing only reverse transcription of poly(A) mRNAs. Then a standard PCR was performed on the mRNA after reverse transcription. The PCR products were visualized on a 1.5% agarose gel stained with ethidium bromide. A single amplification product was seen in positive lanes; the expected size is indicated (in nucleotides) for each product at the bottom of the gel. The control sample was EFB1 mRNA, which includes an intron. Specific PCR of genomic DNA and cDNA exhibits two different products. Reverse transcription after RNase shows no DNA contamination during the process.