Post-transcriptional regulation of gene expression following toxic injury

DNA is the ‘code of life’ storing all the instructions a cell needs, however the information contained in DNA needs to be translated to produce functional proteins. To achieve this an intermediate message, called messenger RNA (mRNA), is generated from the DNA, which is then “read” by large, complex machines called ribosomes, that act as translators travelling along the mRNA reading the instructions of how to build specific proteins. Following exposure to both environmental and therapeutic compounds mRNA can be damaged, and this can result in the misreading of the mRNA by ribosomes or the ribosomes stalling on the mRNA and colliding. Such ribosome collisions are “sensed” by the cell and this activates a number of different cell stress pathway but in particular the ribosome stress response. The aim of this project is to understand more precisely how the ribosome stress response is triggered. This work is important to human health since certain advanced therapeutics contain mRNA, for example the COVID-19 mRNA vaccines. Knowledge from this work will help us to understand how modified mRNAs in vaccines affect such cellular processes, and so can inform the safe design of these new therapeutics.

The overall aim of this programme is to gain a full understanding of the intricate connections between the Ribosome Stress Response (RSR), the Integrated Stress Response (ISR) and associated mRNA translation surveillance pathways. We and others have shown that the RSR and ISR are trigged by direct damage to RNA caused by both environmental (e.g. UVB light) and therapeutic (eg. cisplatin) exposures which result in modification of a range of RNA species. It is now clear that the RSR stimulates an adaptive gene expression program, in part through regulation of transcription. However, data from our laboratory and others suggests that RNA binding proteins (RBPs) involved in controlling post-transcriptional mechanisms are modulated during the RSR, yet there is little information about how the RSR affects post-transcriptional gene expression pathways. We now propose to use genome-wide approaches to determine how the RSR influences mRNA stability, mRNA translation and mRNA localisation in a physiologically relevant context using keratinocytes.
Specific objectives are to i) elucidate the role of RBPs post-transcriptional control during the RSR; ii) identify how post-transcriptional control influence RSR-mediated cell fate decisions; iii) examine how the RSR-ISR axis impacts on the tRNAome. The fundamental information gained in i-iii will be used to understand the impact of the incorporation of modified bases on translational fidelity, as we have shown that incorporation of N1-methylpseudouridine (m1?) into in vitro-transcribed messenger RNA (IVTmRNA) (as used in the COVID19 RNA-based vaccines) results in +1 ribosome frameshifting. Our data strongly suggest that ribosome stalling occurs prior to frameshifting and that the RSR is involved in this process. To gain a full understanding of this process we will: i) identify the translation surveillance mechanisms involved in resolving ribosomes stalled at m1?-dependent stall sites; ii) carry out structural analysis of ribosomes stalled on m1?-modified RNA. This programme will generate a new understanding of the adverse effects of RNA-modification/damage, which hitherto have been unappreciated. This information will be translated to understand the likely impacts of human exposure to agents that activate these pathways. Moreover, these studies will provide key information about the off-target toxicity of RNA-based therapeutics, including mIVTmRNAs and antisense oligonucleotides. Such data are essential to improve the safe design of these advanced therapeutics.