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

几十年来，基因表达一直被认为是一个单向的顺序过程，特别是被核膜分隔的步骤被认为是相互独立的。转录等核过程被认为不受mRNA衰退、翻译和蛋白质降解的影响。现在越来越清楚的是，基因表达的较晚步骤可以影响较早的步骤，导致缓冲变化(内稳态)或加强调节(超调节)。CCR4-NOT(CNOT)复合体似乎在连接核质事件中发挥着重要作用。众所周知，它通过microRNAs 1，2，3，4调节转录，以及介导mRNA的衰退和翻译抑制。在酵母中的实验也表明，CCR4--非复合体将mRNAs的诞生与其衰退3，4结合在一起，但这在哺乳动物细胞中并不是很好的特征。我们最近已经证明，在哺乳动物细胞中，核PolyA尾巴的大小在mRNAs之间存在显著差异，并且核和细胞质Poly(A)尾巴都受CCR4调控--而不是在转录反应期间5。此外，CCR4-NOT亚基的敲除不仅导致更长的聚(A)尾巴和更稳定的mRNA，而且还减少了转录，表明CCR4-NOT在哺乳动物细胞的RNA动态平衡中发挥作用。在这个项目中，您将通过各种方法来修饰CCR4-NOT复合体，包括RNA沉默、CRISPR/Cas9基因操作和使用美国6，7开发的小分子抑制剂进行抑制。使用染色质相关RNA的分离、RNA标记和定量RT-PCR，您将确定要研究的最具信息量的条件，然后进行高通量实验，以确定CCR4-NOT操纵对RNA合成和周转的影响，方法称为SLAM-Seq 8。您将进行广泛的生物信息学分析，以确定感兴趣的mRNA类别及其最可能的转录和转录后调节因子，并使用报告基因和RNA沉默来验证这些假设。这个项目可以更多地转向生物信息学和数学建模，如果你更喜欢这个而不是动手研究的话。你将成为研究CCR4的生物化学家和生物信息学家团队的一员，而不是包括格拉斯哥的马丁·布谢尔的实验室。将有许多关于翻译效率、蛋白质关联和聚(A)尾巴变化的大型数据集可供与您的数据进行比较，从而增加重大发现的机会。BBSRC资助的一个研究项目最近获奖，你可能会在这个项目上与两位博士后密切合作

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