How does the cell distinguish between coding and non-coding RNAs in the cytoplasm?

Neurodegeneration is the progressive loss of function in neurons and is frequently the result of mis-regulation of gene expression. For proper cell function the genetic code needs to be expressed accurately. This relies upon making a copy of the genetic material termed ribonucleic acid (RNA) that moves to the place in the cell where it is needed. Each RNA is bound by a number of protein factors that regulate its activity. Disruptions to RNA-protein (RNP) interactions are highly detrimental to the cell's health. Mutations that upset RNPs can lead to many human diseases including spinal muscular atrophy. To understand such diseases better it is essential to expand our knowledge of how RNAs and proteins interact.
The RNA copy of the gene can either be decoded by the ribosome and a protein made, or the RNA has activity itself without being decoded. We term these RNA that are not decoded, non-coding. The RNAs that are decoded (translated) into protein are coding RNAs. It is essential for the cell to recognise which RNAs should be decoded and which should not. Our current understanding is that these two types of RNA look very much alike.
Many non-coding RNAs are present in neurons and changes in their levels can lead to neuronal cells losing their function, ultimately resulting neurological conditions e.g. Alzheimer's disease. It is therefore important to understand what these non-coding RNAs are doing.
This project will demonstrate which RNAs are coding and non-coding in human neurons. I hypothesise that there are specific protein factors that bind to coding RNAs to mark them for translation and other proteins that mark non-coding RNAs for other activities. I will test this hypothesis and find out which proteins act to distinguish coding and non-coding RNAs, and to discover the biological effect of disrupting these RNA-protein complexes.
For this study, I will use novel sequencing techniques, which I contributed to the development of, to determine which RNAs are non-coding and which are coding in human neurons. Once we know which are which, I will isolate examples of coding and non-coding RNA from cells and look for differences in the proteins that they bind. I propose to perform purification of non-coding and coding RNPs in both Drosophila S2 tissue culture cells and cultured human neurons. To demonstrate the biological importance of these RNPs, I will use genetic tools and create fly strains; disruptions to the RNPs will be assessed in the developing fruit fly.
I will execute experiments both in the model organism, the fruit fly (Drosophila melanogaster) and cultured human neurons. Combining these two models I will look to see if the mechanisms used by cells to differentiate coding and non-coding RNAs are conserved between flies and humans. Fruit flies are ideal for assessing the biological impact of disrupting non-coding RNAs because extensive genetic tools are available. Identifying which RNAs are coding and which are non-coding in human neurons is essential because the RNPs we identify could be important for neuronal function. Characterising cytoplasmic non-coding RNAs in neurons will help further our understanding of how mis-regulation of gene expression in neurons can result in neurological conditions e.g. reduced neural specification. This work will unlock important mechanistic understanding of how cells distinguish between coding and non-coding RNAs, which will advance our understanding of the molecular basis of neurological conditions.

This project will elucidate the mechanisms by which cells distinguish between non-coding and coding RNAs in the cytoplasm. Whilst the majority of our genome is transcribed, only a small fraction is protein-coding. This implies that many non-coding RNAs exist, some of which are similarly processed to mRNAs, termed long non-coding RNAs (lncRNAs). The majority of characterised lncRNAs are nuclear but recent ribosome profiling results have revealed that many are translated. Although controversial, it is clear that numerous lncRNAs are cytoplasmic, polysome-associated and translated. The line between coding and non-coding has become blurred and some lncRNAs are really mRNAs. I hypothesise that cells can differentiate between coding and non-coding RNA, by virtue of the different proteins bound. I will identify what these molecular markers are and investigate their biological importance.
LncRNAs are enriched in neuronal tissues and several have been found associated with neurological conditions e.g. Alzheimer's disease. An inability to differentiate between coding and non-coding RNAs will have profound effects on cellular health. To better understand the molecular basis of neuronal disease it is vital that we appreciate how coding and non-coding RNAs are distinguished by the cell and the role of cytoplasmic lncRNAs in regulating gene expression.
I developed Poly-Ribo-Seq to identify regions of active translation with an improvement to ribosome profiling and discovered which lncRNAs are translated in Drosophila S2 cells. Using cultured human neurons, I will now employ Poly-Ribo-Seq to identify which RNAs are translated and which are not in human neurons. I will purify lncRNPs and mRNPs from Drosophila and human neurons to identify which proteins bind to the different RNA pools. This will demonstrate the molecular differences between lncRNAs and mRNAs and provide novel and timely insight into how mis-differentiation of RNA populations can promote neurological disease.

This proposal will dissect the molecular mechanisms by which cells distinguish between coding and non-coding RNAs in the cytoplasm and whether this machinery is conserved between Drosophila and humans. We will profile RNA populations in cultured human neurons and identify protein complexes, which help define these populations. This research is relevant to the work of neurogenerative diseases and how lncRNAs may contribute to their progression.

To ensure that our research reaches its impact potential we will a) publish in high-quality open-access journals, b) present our work at international, national and local conferences, c) develop internet based tools to communicate with scientists and the public (twitter and website), d) make our data-sets available to use and download by others (NCBI), e) take part in outreach activities in the Southampton community, e) provide high quality training to researchers, f) develop collaborations in the inter-disciplinary Institute for Life Sciences and g) discuss technical advances within the genomics field, h) interact with Next Generation Sequencing technology companies to develop novel reagents.

This research will quickly benefit academic scientists in the local RNA field based on our groundbreaking research and then the wider international field as work is published. Scientists in other disciplines at Southampton will benefit from our research by interactions and collaboration, which will start as we integrate into the Southampton community e.g. join the interdisciplinary nucleic acids network and will continue into the future. Training of research staff will have a large and rapid impact on their skills and career development, and their own impact on the field will continue for years to come.

The relevance of this research to the study of neurological diseases means our results in cultured human neurons will influence scientific enquiry by neurobiologists and health care professions involved in personalised medicine, in the mid-term. RNAs we profile may be ideal candidates for diagnostic and prognostic biomarkers. Advances made based on our findings on mutant RNA phenotypes and mechanisms of recognition will have effects on society through medicine in the long-term. In the future data-sets from my genomic experiments could lead to RNA development of therapeutics by pharmaceutical companies. Potential RNA inhibitors could provide large economic and societal impact in the future. Working with the Research and Innovation Centre at Southampton will facilitate these potential outcomes of our research. The long-term aim is that understanding more about the difference between coding and non-coding RNAs will help treat human disease.
My research and scientific expertise will effect the local public especially children and young adults as I will participate in outreach activities in the community. In these endeavours I will communicate my science to non-scientists and encourage students to study science and pursue scientific careers.

In summary many different sectors of society will be affected by our research and I have developed ways in which to maximise this and measure the impact over time.