Does the CNOT tie the ends? Investigating the circularity of gene expression.

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For decades, gene expression has been thought of as a one-way sequential process, in which especially the steps separated by the nuclear membrane were presumed to be independent of each other. Nuclear processes such as transcription were thought to not be influenced by the mRNA decay, translation and protein degradation. It is now becoming clear that the later steps in gene expression can influence the earlier ones, leading to buffering of changes (homeostasis) or enhancement of regulation (hyper-regulation). The CCR4-NOT (CNOT) complex appears to play a major role in coupling nuclear and cytoplasmic events. It is known to regulate transcription, as well as mediate mRNA decay and translational repression by microRNAs 1,2 3,4. Experiments in yeast also indicate the CCR4-NOT complex couples the birth of mRNAs to their decay 3,4, but this is not well characterised in mammalian cells. We have recently shown that nuclear poly(A) tail sizes differ markedly between mRNAs in mammalian cells and that both nuclear and cytoplasmic poly(A) tails are regulated by CCR4-NOT during a transcriptional response5. Moreover, knockdown of CCR4-NOT subunits does not only lead to longer poly(A) tails and more stable mRNA, but also reduces transcription indicating a role for CCR4-NOT in RNA homeostasis in mammalian cells. In this project you will modify CCR4-NOT complexes by a variety of methods, including RNA silencing, CRISPR/Cas9 gene manipulation and inhibition with small molecule inhibitors developed by us 6,7. Using isolation of chromatin associated RNA, labelling of RNA and quantitative RT-PCR you will identify the most informative conditions to study and then conduct high throughput experiments to determine the effect of CCR4-NOT manipulation on the synthesis and turnover of RNA using a modification of a method called SLAM-Seq 8. You will do extensive bioinformatic analysis to determine mRNA classes of interest and their most likely transcriptional and post-transcriptional regulators and test these hypotheses using reporter genes and RNA silencing. This project can be shifted more towards bioinformatics and mathematical modelling if you prefer this to the hands-on research.You will be part of a team of biochemists and bioinformaticians investigating CCR4-NOT, including the laboratory of Martin Bushell in Glasgow. There will be many other large datasets on translational efficiency, protein association and poly(A) tail changes available for comparison with your data, enhancing the chance of a major discovery. A BBSRC funded research project has recently been awarded and you are likely to work closely with the two postdocs on this project