Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism used by many viral and cellular mRNAs. PRF occurs at a heptanucleotide slippery sequence and is stimulated by a downstream RNA structure, most often in the form of a pseudoknot. The utilization of −1/ +1 PRF to produce proteins encoded by the −1/+1 reading frame is wide-spread in RNA viruses, but relatively rare in cellular mRNAs. In human, only three such cases of −1 PRF events have been reported, all involving retroviral-like genes and protein products. To evaluate the extent of PRF utilization in the human transcriptome, we have developed a computational scheme for identifying putative pseudoknot-dependent −1 PRF events and applied the method to a collection of 45,000 human mRNAs in the NCBI RefSeq database. Using core program PKscan, we have performed a large-scale search for putative pseudoknot dependent PRF. In addition to the three reported cases, our study identified more than two dozen putative −1 PRF cases. The genes involved in these cases are genuine cellular genes without a viral origin. Moreover, in more than half of these cases, the frameshift site locates far upstream from the stop codon of the 0 reading frame, which is nonviral-like.Using dual luciferase assays in HEK293FT cells, we confirmed that the −1 PRF signals in the mRNAs of CDK5R2 and SEMA6C are functional in inducing efficient frameshifting. We also present evidence to show that the mRNA of human inorganic pyrophosphatase 1 (PPA1) harbors functional cis-acting signals for +1 PRF. The consequence of frameshifting is the production of a longer PPA1 protein in which the C-terminal 25 residues of normal PPA1 are replaced by 68 residues translated from the +1 reading frame. To the best of our knowledge, the human PPA1 mRNA is the only other mammalian cellular mRNA known to date to utilize the +1 PRF mechanism, besides the antizyme mRNAs. Results from the studies on the involvement of PPA1 in tumorigenesis suggest that PPA1 is a potential prognostic biomarker for certain cancers, and strategies for PPA1 down-regulation may have therapeutic potential for the treatment of cancers. This study also recognized a new pseudoknot structure in SARS-CoV that has been validated as a cis-acting regulation factor in viral frameshifting event, referred to 'intertwined double pseudoknots’. They are present at the −1 PRF site in SARS-CoV-1/2 and many other coronaviruses. An even larger scale analysis on transcriptome-wide study in all available human mRNAs identified many of them have the potential to form the same structure, which may involve in regulation of translation initiation, or mRNA stability. Finally, preliminary design of CRISPR-inspired strategy to induce -1 ribosomal frameshifting by trans-acting factors was verified to be functional. Our findings have significant implications in expanding the repertoire of the PRF phenomenon in both human and viral mRNAs and the protein-coding capacity of the human transcriptome.

Pseudoknots are essential for the functioning of many small RNA molecules. In addition, viral RNAs often exhibit pseudoknots that are required at various stages of the viral life-cycle. Techniques for detecting evolutionarily conserved, and hence most likely functional RNA pseudoknots, are therefore of interest. Here we present an extension of the alidot approach that extracts conserved secondary structures from a multiple sequence alignment and predicted secondary structures of the individual sequences. In contrast to purely phylogenetic methods, this approach yields good results already for small samples of 10 sequences or even less.