Model of protein translation and quantitative assay identify dozens of novel slippery sites causing ribosomal frameshifting

Cellular life critically depends on accurate translation of mRNAs into proteins. However, translation is an imperfect process, and the ribosome is known to make a variety of errors, termed phenotypic mutations. Phenotypic mutations include single amino acid substitutions, Stop codon readthrough, or ribosomal frameshifting, where the ribosome switches from one reading frame to another during translation. Ribosomal frameshifting in particular is implicated in human health, as many viruses, including HIV and Sars-CoV2, use it and depend on it. In addition, recent evidence suggests that frameshifting occurs during translation of the Sars-CoV2 mRNA vaccines. Mechanistically, frameshifting is generally dependent on signals in the mRNA sequence, so-called "slippery sites". While the biochemical mechanisms of frameshifting have been studied in detail, few attempts have been made to identify frameshifting sites independent of known cases and at high throughput.

We developed a mechanistic model of translation errors purely based on the mRNA sequence and the cell"s tRNA pool that predicts frameshifting sites. Using a reporter protein construct that allows frameshift quantification by fluorescence and mass spectrometry, we tested the model"s predictions in vitro. Our assay identified dozens of 10-mers that cause frameshifting by more than 10%, both in the -1 and +1 direction. Considering that all of the tested sequences occur in real genomes, this indicates that frameshifting outside of viruses is more common than previously assumed. In addition, our model predicts -1 and +1 frameshifting with an average precision of >80%, both on the experimental library and a larger dataset of known frameshift motifs and non-frameshifting sequences.

Using the experimental data and the model"s predictions, we are now scanning genomes for frameshifting sites. This will allow us to assess the baseline levels of frameshifting and thus its impact on an organism"s fitness. In addition, we are analyzing the putative protein products of these frameshift sites in terms of their conservation, structure and family membership to identify novel functional cases of frameshifting.