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，特别是被核膜分开的步骤被认为是相互独立的。转录等核过程被认为不受mRNA衰变、翻译和蛋白质降解的影响。现在越来越清楚的是，基因表达的后期步骤可以影响早期的步骤，导致变化的缓冲（稳态）或调节的增强（超调节）。CCR4-NOT （CNOT）复合物似乎在细胞核和细胞质事件的耦合中起主要作用。已知它可以调节转录，并通过microrna 1、2、3、4介导mRNA衰变和翻译抑制。酵母实验也表明，CCR4-NOT复合体将mrna的产生与它们的衰变结合在一起，但这在哺乳动物细胞中并没有很好地表征。我们最近的研究表明，哺乳动物细胞中不同mrna的核多聚(A)尾的大小存在显著差异，并且在转录应答过程中，核多聚(A)尾和细胞质多聚(A)尾都受到CCR4-NOT的调控5。此外，敲低CCR4-NOT亚基不仅会导致更长的聚(A)尾和更稳定的mRNA，而且还会减少转录，这表明CCR4-NOT在哺乳动物细胞中RNA稳态中的作用。在本项目中，您将通过多种方法修饰CCR4-NOT复合物，包括RNA沉默，CRISPR/Cas9基因操作和我们开发的小分子抑制剂抑制6,7。通过分离染色质相关RNA，标记RNA和定量RT-PCR，您将确定最具信息量的条件进行研究，然后进行高通量实验，以确定CCR4-NOT操作对RNA合成和转换的影响，使用一种称为SLAM-Seq 8的方法进行修改。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沉默来测试这些假设。这个项目可以转向更多的生物信息学和数学建模，如果你喜欢这比动手研究。你将成为研究CCR4-NOT的生物化学家和生物信息学家团队的一员，其中包括格拉斯哥的Martin Bushell实验室。将有许多其他关于翻译效率、蛋白质关联和多聚(A)尾部变化的大型数据集可与您的数据进行比较，从而增加重大发现的机会。最近有一个BBSRC资助的研究项目被授予，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