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

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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.