Identification of functional, endogenous programmed -1 ribosomal frameshift signals in the genome of Saccharomyces cerevisiae - PubMed (original) (raw)

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

Jonathan L Jacobs et al. Nucleic Acids Res. 2007.

Abstract

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.

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Figures

Figure 1

Figure 1

Typical −1 PRF signals consist of a heptameric slippery site that fits the motif N NNW WWH (spaces indicate zero frame codons), a short spacer region of 5–12 nt, and an mRNA pseudoknot with two stem and three loop regions (S1, S2 and L1–L3, respectively). See Materials and Methods for the pseudoknot motif criteria used in this study.

Figure 2

Figure 2

Scatterplot of MFE values (predicted using pknots, (24) versus zR scores for 10 340 candidate −1 PRF signals demonstrates the weak correlation between these two feature statistics (see Supplementary Table 2B). The red diamonds and associated labels indicate the location and parental gene of nine sequences empirically tested for frameshifting. The hypothetical distributions were created using summary statistics from Supplementary Table 2B.

Figure 3

Figure 3

Nine examples of candidate −1 PRF signals chosen to generally represent the diversity of features present in the PRFdb. Gene names are shown with RNA sequence and corresponding CDS nucleotide start and stop locations. The predicted structure is shown for each empirically tested candidate signal. See Supplementary Table 3 for statistical data.

Figure 4

Figure 4

Measurement of −1 PRF efficiency for nine candidate signals. (A) High-efficiency frameshifting including the frameshift signal from the endogenous yeast L-A virus. (B) Medium- and low-efficiency frameshifting including the sequence from the FKS1 gene that did not promote −1 PRF above background levels. The parental genes of each candidate signal are indicated with the percentage of −1 PRF efficiency as was measured using a dual-luciferase reporter assay system (36,37).

Figure 5

Figure 5

(A) The CDS of S.cerevisiae is not prone to lengthy out-of-frame translation. The relative positions of candidate −1 PRF signals from the start codon of each ORF compared to the expected overall change in peptide length if a frameshifting event were to occur. (B) Fraction of ORFs containing high probability −1 PRF signals represented as mRNAs stabilized in strains deficient in NMD (_upf1_Δ, _upf2_Δ or _upf3_Δ) (60), No-go decay (_xrn1_Δ or _dcp1_Δ) (60), or having half-lives less than the yeast transcriptome average (t1/2) (70).

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