KnotInFrame: prediction of -1 ribosomal frameshift events

Theis, Corinna; Reeder, Jens; Giegerich, Robert

In the study of (5), 1679 candidates were classified as strong candidates, due to a good z-score and a low-MFE value. These candidates were made available by the authors in their PRFdb [(28), http://dinmanlab.umd.edu/ prfdb/]. First, we asked what portion of those 1679 candidates were also predicted by our pipeline. On the first glance, the result was astonishing: only 257 sites were confirmed by our analysis, i.e. they were predicted within one of the first five ranks for each ORF. By visual examining of some of the missed candidates the reason was immediately obvious. The predicted structure in the PRFdb often does not resemble the pseudoknot frameshift consensus. Instead, we find virtually all possible other structures, such as a single hairpin, a chain of hairpins, bifurcated structures or complex pseudoknot structures. This supports our main criticism of the approach of Jacobs et al.: the candidate sequences have the base-pairing potential to build a pseudoknot specified by the (RNAmotif) consensus, but for most sequences an alternative structure is more stable. This alternative structure is then used for the z-score computation and stored in the PRFdb. In contrast, our approach explicitly folds the consensus pseudoknot and the alternative structure and assigns to candidates with a more stable alternative structure a negative Á and consequently a low rank. Therefore, we filtered the list of strong candidates in the PRFdb for structures containing at least one pseudoknot and obtained 163 candidates. Next, we tested if our predictions are supported by Jacobs et al. We created a list of our 100 strongest candidates by setting a threshold of Á ! 0:08. Again, only a small portion (13) of these predictions are supported by the PRFdb. The reasons for this are manifold: first, Jacobs et al. are using a more stringent consensus, which allows only 1-3 bases in loop 1. Second, the folding program used in the creation of the PRFdb (pknots) uses an outdated energy model, while KnotInFrame uses the most up to date energy model for nested structures and an adapted model for pseudoknots. In consequence, it happens that a stable structure reported by KnotInFrame is rejected in the PRFdb due to a possibly worse energy reported by pknots. Remarkably, the maintainers of PRFdb started to include another RNA folding algorithm [NUPACK (29) ] into the pipeline, which uses an energy model close to ours. Database entries derived from NUPACK analysis seem to be more consistent with our results. However, we cannot quantify the improvement, since the inclusion of NUPACK seems to be work in progress and only parts of the database are currently reprocessed.