Slip-Sliding the Frame: Programmed −1 Frameshifting on Eukaryotic Transcripts

Abstract

The basic mechanisms of mRNA translation are ubiquitous among all organisms, in that the accurate decoding of triplet codon sequences programs serial amide linkages of amino acid residues by ribosomal complexes. In general, the fidelity of this process is dependent on both accurate recognition of mRNA codons by aminoacyl tRNAs and maintenance of the corresponding open reading frame. However, in a growing number of cases, deviations from this triplet codon rule are observed, indicating that the information content of an mRNA to encode protein may extend beyond its primary structure. These cases, collectively referred to as translational recoding (for review, see Gesteland and Atkins 1996), present exceptions to the venerable genetic code and are typically subdivided among three primary mechanisms: (1) frameshifting, in which translating ribosomes are induced to slide one nucleotide forward or backward at a distinct point in the transcript, with protein synthesis then continuing in the +1 or 11 reading frame, respectively; (2) alternative codon usage, where stop codons are not interpreted as sites of translational termination but, rather, encode an amino acid residue; and (3) translational bypassing, where the translational machinery traverses a gap in the mRNA coding sequence yet yields a single polypeptide chain. Programmed 11 ribosomal frameshift signals are among the most extensively characterized of these translational recoding phenomena (for review, see Brierly 1995; Dinman 1995; Farabaugh 1996). Although current examples are largely limited to viral systems, some bacterial 11 frameshifting events have also been documented (Blinkowa and Walker 1990; Tsuchihashi and Brown 1992; Chandler and Fayet 1993; Engelberg-Kulka and Schoulaker-Schwarz 1994). At present, no examples of eukaryotic mRNAs exhibiting 11 frameshift activity have been reported. However, a number of viruses infecting eukaryotic cells utilize programmed 11 ribosomal frameshifts, demonstrating that the cis elements involved in the frameshifting process are operational in eukaryotes. In viral systems, the efficiency of frameshifting is an essential determinant of the stoichiometry of synthesized viral protein products, which must be rigidly maintained for efficient propagation of the virus (Brierly 1995 and references therein; Dinman and Wickner 1995). In work presented in this issue, (Hammell et al. 1999), a bioinformatic approach was used to screen prokaryotic and eukaryotic DNA sequence databases for potential 11 frameshift signals. Because of the complexity of 11 frameshift sites and the sequence variability observed among these elements in nature, a multicomponent search algorithm was required to identify candidate sites from bulk database entries. This was made possible in part by the plethora of information available describing structural features of frameshift signals, much of which is described in Hammell et al. (1999) and elsewhere (Brierly 1995; Chen et al. 1996; Marczinke et al. 1998). This strategy represents a marked contrast to many examples of database searching, which typically involve a single motif or are limited to primary structure parameters (Fickett 1996; Altschul et al. 1997; Aravind and Landsman 1998; O’Neill 1998). Database searches using this compound algorithm for 11 frameshift sites yielded a host of potential signals in all databases tested, including eukaryotic sequences, with frequencies significantly higher than found in random sequence populations. In a number of cases, frameshift signals were conserved in homologous mRNAs from different species. A convincing argument for the validity of these searches was provided by the functional demonstration of 11 frameshifting activity for two selected signals (from Saccharomyces cerevisiae RAS1 and human CCR5 mRNAs) in a recombinant assay system. Furthermore, in four cases, potential 11 frameshift signals colocalized to sites of mutation linked to heritable diseases in humans, raising the possibility that 11 frameshift activity may be a physiologically relevant component of regulated gene expression in humans. Modifications of the parameters employed in this search algorithm may yet reveal additional candidate frameshift signals, as the potential for 38-RNA pseudoknot formation was a constraint placed upon their selection in this study. An RNA pseudoknot is not requisite for 11 frameshifting at the gag–pol overlap of HIV-1 (Jacks et al. 1988), for example, which only contains a weak 38-stem–loop structure in vivo (Parkin et al. 1992). The identification of functional 11 frameshift signals in chromosomally encoded eukaryotic mRNAs raises several interesting questions for further investigation. First, it will be important to determine if ribosomal frameshifting occurs in vivo in the context of these transcripts. If so, what is the function of the frameshift event? A ribosomal frameshift may serve to generate alternate protein isoforms, with common aminoCorresponding author. E-MAIL gbrewer@wfubmc.edu; FAX (336) 7169928. Insight/Outlook