In the framework of this project we have performed the following work.
First, we have developed a software application that lets us make inferences about the movement of the ribosome at single nucleotide resolution, uncovering general and gene- and codon- specific patterns, based on datasets produced by capturing and sequencing mRNA degradation intermediates. The application is written in python and is called fivepseq (http://pelechanolab.com/software/fivepseq/ ). We have shown the wide range of applications of these analyses across dataset types and organisms. We have also used fivepseq to identify degradations frameshifts in yeast, and published these results in NAR Genomics and Bioinformatics [1].
Next, we investigated ribosome dynamics in degrading mRNAs in yeast, under various types of stress, including nutrient deprivation (poor media) and salt stress. Indeed, we observed a clear frameshift in poor nutrient conditions (Figure 1). This was further aggravated upon addition of salt. Such an effect could be a consequence of ribosome “slippage”. The latter should in theory produce small amounts of shifted peptides, which our collaborators have confirmed with mass-spectrometry analysis. Finally, to confirm that the frameshift indeed leads to activation of nonsense-mediated decay (NMD) machinery, we have assessed mRNA stability in strains with or without functional NMD. Indeed, while the NMD dysfunction did not affect mRNA stability in normal conditions, it did increase mRNA stability in stress. These results will be finalized in another manuscript by the end of this year where the MCIF funding will be acknowledged.
While 5PSeq is straightforward to apply in yeast, the mRNA degradation is a much more complex process in higher eukaryotes, including humans. We have performed modifications to the fivepseq application to remove technical and biological noise in these datasets, understand translational state in human cells. With further improvement of the experimental pipeline to produce clean 5PSeq data from low input RNA in human cells (by a postdoc in the host lab), we hope to achieve novel applications in humans.
Moving beyond the state of the art, we discovered that coupling between translation and degradation processes happens not only in eukaryotes, but also in bacteria. This discovery enabled us to expand the applications of the 5PSeq technology to assess translational states in bacteria with minimum experimental perturbations and identify fast bacterial responses to antibiotics. This is possible, because many antibiotics are targeting the ribosome, and eventually affect the patterns of generation of mRNA degradation intermediates. Furthermore, our technology enables assessment of bacterial response to treatment in complex mixtures of microbiomes, at single species and single nucleotide resolution. To our knowledge, this is the only technology so far that enables such measurements without the need to isolate and culture individual species, with minimum experimental perturbations. These results are published in a preprint [2] and will be submitted for publication by the end of this year.
I have also been involved in a few collaborations in Sweden, namely with labs of Vasili Hauryliuk and Joanna Rorbach.
