Periodic Reporting for period 1 - TERMINATOR (Ribosomal frameshifts as a novel mechanism to control RNA turnover in stress)
Berichtszeitraum: 2019-08-01 bis 2021-07-31
We were interested to investigate cellular response to changes in the environment, by watching how the cell degrades its mRNAs. mRNAs are used to make proteins, and the type of mRNA produced will affect the types of proteins synthesized. The cell continuously synthesizes and degrades these molecules. When a cell is exposed to stress, it will need to synthesize other types of proteins, and, hence, it should get rid of the old mRNAs very fast. We suggested a possible mechanism that could lead to such a clean-up, associated with the movement of ribosomes - the machinery that synthesizes proteins. The ribosomes move along the mRNA molecule one codon at a time, and in each movement incorporate a new amino acid to the growing protein chain. However, if the ribosome “slips” one nucleotide, i.e. when its frame is shifted, it will start producing a wrong protein and will terminate at a premature termination codon (PTC). The latter is usually noticed by the nonsense-mediated decay (NMD) machinery. NMD normally degrades erroneous transcripts. However, we hypothesized that the cell uses NMD to also degrade normal mRNAs as a response to stress, via genome-wide ribosomal frameshifts. Previous work had shown mRNA degradation and ribosome movement are coupled, and that degradation intermediates are a “living trace” of how the ribosome moves in the cell. Therefore, we used a novel technology - 5PSeq - to look into these degradation intermediates and make inferences regarding movement of the ribosomes on those mRNAs that are getting degraded.
Understanding how the cell responds to stress and treatment is an important milestone in science to investigate mechanisms of disease development and efficacy of drug treatments. mRNA degradation is only one of the processes that can provide better assessment of response mechanisms. It has, however, the advantage of showing fast cellular responses, as responses that occur at transcriptional, epigenetic or genetic levels occur at longer timescales. The overall objective of this work was to look at the mRNA degradation intermediates with a new technology called 5PSeq, and develop novel data analysis methods to investigate cellular response to stress.
In the course of this project, we also expanded this work by investigating that same process in bacteria. By doing so, we discovered that mRNA degradation is coupled with the process of translation not only in eukaryotes, but also in prokaryotes. This enabled us to develop a technology that opens novel avenues for understanding bacterial response to antibiotic treatment, enabling more efficient diagnostics and drug discovery workflows.
Understanding how the cell responds to stress and treatment is an important milestone in science to investigate mechanisms of disease development and efficacy of drug treatments. mRNA degradation is only one of the processes that can provide better assessment of response mechanisms. It has, however, the advantage of showing fast cellular responses, as responses that occur at transcriptional, epigenetic or genetic levels occur at longer timescales. The overall objective of this work was to look at the mRNA degradation intermediates with a new technology called 5PSeq, and develop novel data analysis methods to investigate cellular response to stress.
In the course of this project, we also expanded this work by investigating that same process in bacteria. By doing so, we discovered that mRNA degradation is coupled with the process of translation not only in eukaryotes, but also in prokaryotes. This enabled us to develop a technology that opens novel avenues for understanding bacterial response to antibiotic treatment, enabling more efficient diagnostics and drug discovery workflows.
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.
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.
Our project has first of all produced fundamental knowledge, showing the possibility that ribosome frameshift induced mRNA degradation is a potential mechanism controlling cellular response to stress. In addition, we have shown that the processes of translation and mRNA degradation are co-occurring in prokaryotes – something that was not known before.
In addition, we have shown the huge potential of our meta-degradome technology for translation towards diagnostics and antibacterial drug development pipelines. This will increase our ability for surveillance of antibiotic resistance, which is a major challenge in the 21st century, as more and more bacterial infections become deadly once again. We will continue working on our technology to turn it in as a solution to this exacerbating problem.
Our project has produced one published paper (fivepseq application), one preprint (meta-degradome technology) and a manuscript in preparation (frameshift mediated mRNA degradation). The novel meta-degradome technology has been presented in several workshops and conferences, including by the applicant at the “Science talks'' conference at SciLifeLab in Solna, in June 2021. By the end of the project, we formed a company to further develop the meta-degradome technology and increase the socio-economic impact of our work..
In addition, we have shown the huge potential of our meta-degradome technology for translation towards diagnostics and antibacterial drug development pipelines. This will increase our ability for surveillance of antibiotic resistance, which is a major challenge in the 21st century, as more and more bacterial infections become deadly once again. We will continue working on our technology to turn it in as a solution to this exacerbating problem.
Our project has produced one published paper (fivepseq application), one preprint (meta-degradome technology) and a manuscript in preparation (frameshift mediated mRNA degradation). The novel meta-degradome technology has been presented in several workshops and conferences, including by the applicant at the “Science talks'' conference at SciLifeLab in Solna, in June 2021. By the end of the project, we formed a company to further develop the meta-degradome technology and increase the socio-economic impact of our work..