Novel mechanisms controlling the cellular stress response

A protective stress response (SR) is generated in all cells following exposure to physiological stressors such as elevated temperatures, ischaemia, osmotic shock and many other stressors. The response is characterized by the suppression of normal cellular transcription and translation and the synthesis of heat shock proteins (HSPs) that protect cells from damage largely by preventing the abnormal folding and aggregation of proteins and by mediating anti-apoptotic effects. The SR in mammals is under the control of heat shock transcription factors (HSFs) and following a stress they mediate: (i) the transcription of protective heat shock protein; (ii) drive the expression of the long non-coding RNAs Sat III encoded on non-mapped peri-centromeric regions of human chromosomes. Under stress conditions Sat III transcripts of varying length are transcribed and form scaffolds for specific transcription factors and RNA binding proteins and these sites of accumulation are termed nuclear stress bodies (nSBs) and are involved in mediating the cessation of transcription and control of splicing. The stress response of non-dividing neurones has been reported to be cell-type specific and the kinetics and level of expression of HSPs vary according to the type of stress. Importantly, altered HSF1 function is associated with ageing, cancer and many neurodegenerative diseases and suggests an altered HSR may contribute to disease aetiology. We have found previously undocumented important roles for the scaffold attachment factor B (SAFB) family of RBPs in coordinating the stress response in neuronal and non-neuronal populations of cells. SAFB1 and SAFB2 are ubiquitously expressed and found at very highly levels in the human brain. We have previously shown they coordinate the expression of coding and non-coding genes and that they are involved in alternative spicing. We have further novel findings showing that SAFB1 regulates the transcription/ stability of Sat III RNA and its paralogue SAFB2 translocated to Sat III/nSBs with the same kinetics as HSF1. In addition, both SAFB1 and SAFB2 are arginine methylated in domains known to mediate both protein-RNA and protein-protein interactions. RNA binding proteins (such as SAFB1/2) involved in regulating the stress response and found in stress bodies have also been shown to implicated in the ageing process and in the aetiology of human neurodegenerative disease.
We propose that SAFB1 and SAFB2 are key components governing the cellular response to stress, and that their functions in protein complexes may be regulated by post-translational arginine methylation. Specifically, we hypothesise they are required for Sat III transcription and the recruitment of RBPs to nSBs which in turn Sat govern the cellular stress response. The specific research objectives are: 1. Identify the roles SAFB1 and SAFB2 play in regulating Sat III transcription and nSB formation. 2. Define the role arginine methylation plays in governing the functions of SAFB1 and SAFB2. 3. Identify novel SAFB1 and SAFB2 interaction partners that function under stress and non-stress conditions. 4. Characterise the role SAFB proteins play in regulating Sat III lncRNA transcription and the formation of nSBs in human neurones.

The outcome of this study will be to elucidate the mechanisms by which SAFB (and other RNA binding proteins) regulate Sat III transcription and nSB formation and thereby the cellular stress response. Hence novel insights will made into the processes that underpin the neuronal and non-neuronal cell response to environmental stress, govern ageing, cancer and underpin a number of human neurodegenerative disease conditions.

Cells can elicit a stress response characterized by the suppression of normal cellular transcription and translation and the synthesis of heat shock proteins (HSPs) that buffer them from damage. HSF1 governs the transcription of HSP genes and also of human satellite III RNA transcripts that form the scaffold for nuclear stress bodies (nSBs). Recently proteins known to interact with SAFB1 were shown to play important roles in recognising non-coding RNAs (similar to Sat III transcripts) and to have a role in regulating the cellular response to stress. We have novel findings showing that SAFB2 is recruited to nSBs at the same time as HSF1 and much earlier than SAFB1, and that SAFB1 regulates Sat III transcription/stability. In addition, SAFB1 proteins are arginine methylated in domains that determine their interactions with nucleic acids and protein partners. Based on these observations, we hypothesise that SAFB1 and SAFB2 are key components of the protein complexes that govern the cellular response to stress. To test this hypothesis, we will use a combination of biochemical, molecular, imaging and proteomic techniques to: (i) investigate whether SAFB1 and SAFB2 regulates the stress response via a direct or indirect interactions with Sat III transcripts; (ii) investigate how the methylation of SAFB proteins governs their functions; (iii) identify novel SAFB1 and SAFB2 interaction partners involved in regulating the stress response; (iv) characterise the stress response in human neurones.

The generation of new biomedical knowledge and scientific advancement will be significant immediate impacts. This research will benefit: (i) local researchers interested in the control of gene expression, cancer and working with neurons derived from human pluripotent stem cells; (ii) the academic molecular biology and neuroscience research communities; (iii) those in the field of educational science; (iv) the research staff employed on the grant will benefit from training in stem cell biology (v) members of the general public with an interest in cellular and brain function and disease; (vi) Industrial partners.
(i, ii & iii). The research will be disseminated through peer-reviewed journals within the standard timescale. The research will be presented to the scientific community at national and international conferences. The impact of the research will also be increased once disseminated to our existing collaborators (and upon the establishment of new collaborations): these include collaborations within Bristol (School of Physiology and Pharmacology, School of Clinical Sciences, Southmead Hospital), Cardiff University Medical School, the Heath Hospital and our extensive network of industrial collaborators. The potential long-term scientific opportunities are most closely allied with biomedical impacts. The stress response protects cells during their lifetime, becomes attenuated with age and an altered stress response is linked to human diseases such as cancer and neurodegeneration. Identifying novel mechanisms by which the stress response is regulated will therefore be of interest to a very broad range of researchers and to the pharmaceutical industry. The applicants have experience in presenting the research to the Media, scientific community and wider public audiences including school children through public engagement seminars organised by Bristol Neuroscience and via local Bristol Cancer charities.
(iv) - Staff - Dr Scott is an experienced molecular biologist who will benefit from a training in stem cell biology, namely the differentiation of neurons from human pluripotent stem cells. Dr Scott will also communicate our findings to scientific and general audiences through public engagement opportunities; team working and networking.
(v) - The public - findings from this project in the short term will allow us to explore the relationship between the mechanisms governing gene expression and how altered function could lead to the formation of human neurological disease. The brain is a very important organ, commanding special interest from the public, because it holds our memories and governs our behaviour and perceptions. My laboratory gave talks and run workshops at the Bristol Neuroscience festival (Oct 2013-2017) and this great event was extremely popular to a diverse public audience. Other neuroscience activities led by Bristol researchers - e.g. during Brain Awareness Week (a biennial hands-on research festival with a total audience of 4,700) - are equally popular with public audiences, as are public talks on neuroscience topics held regularly by Bristol Neuroscience (BN) http://www.bristol.ac.uk/neuroscience.

(iv) The pharmaceutical industry is always evaluating potential new therapeutic avenues to explore. Understanding how a cell responds to stress impacts on a number of human disease areas, for example drugs that activate the stress/ heat shock response are used in the treatment of Cancer and modifiers of the stress response are being evaluated in the treatment of neurodegenerative disease. We have strong links with Industry, e.g. Takeda UK (now Cerevance Ltd.) and OxfordBiomedica and together with Bristol Research Enterprise and Development (RED) .we will maximise commercial potential development opportunities. Regular meetings with representatives from RED Innovation will also be scheduled to identify such opportunities