Studying RNA metabolism at single nucleotide resolution; the role of RNA modification in transcript processing.

最近的研究表明，RNA修饰是一个重要的生物学过程，与RNA的命运有关，并与其他分子相互作用。RNA修饰被认为在多种细胞、发育和疾病过程中发挥作用。据估计，有超过170种不同的RNA修饰，其意义尚未被理解。该项目涉及使用细胞系来验证和理解RNA修饰在转录物加工中的作用的实验，其中特定的转录物在降解之前保留在细胞核内。我们已经从患有强直性肌营养不良（DM）的患者中建立了细胞系，该疾病与进行性肌无力和消瘦以及认知能力下降和白内障有关。这种情况的许多方面都与过早衰老有关。DM是显性遗传的，由转录的扩增DNA序列（CTG）引起，但突变的RNA被困在细胞核中，在那里它形成不同的核糖核灶，可以通过原位杂交观察到。最近的数字PCR数据表明，与野生型转录本相比，突变体转录本以7倍的过量存在。这种转录物被标记为通过一种尚不清楚的机制降解，但可能涉及RNA修饰。该项目涉及基于独特细胞系的研究，使用互补珠下拉捕获突变RNA，并通过一系列分子，细胞和化学方法进行分析。该项目的测序部分将涉及化学学院制备的RNA修饰标准品。除了上述细胞系之外，该项目还将涉及CRISPR Cas9基因组工程，以在DNA重复扩增上游引入诱导型启动子，从而可以通过多西环素诱导来控制突变体RNA的表达。这将允许产生更高水平的突变体转录物用于捕获和分析。将使用一系列技术分析重复扩增RNA，包括超分辨率显微镜、纳米孔测序、薄层色谱和质谱。该项目的目的是研究单核苷酸分辨率的RNA修饰，以深入了解支撑RNA代谢的分子过程。

Recent studies indicate RNA modification is an important biological process, relevant to RNA fate, and interactions with other molecules. RNA modification is thought to play a role in multiple cellular, developmental and disease processes. It is estimated that there are more than 170 different RNA modifications, and their significance has yet to be understood. This project involves experiments to characterise and understand the role of RNA modification in transcript processing using cell lines in which specific transcripts are retained within the nucleus prior to degradation. We have established cell lines from patients with a condition called myotonic dystrophy (DM) which is associated with progressive muscle weakness and wasting, in addition to cognitive decline and cataracts. Many aspects of the condition are associated with premature ageing. DM is dominantly inherited, caused by an expanded DNA sequence (CTG) which is transcribed but the mutant RNA is trapped in the nucleus where it forms distinct ribonuclear foci that can be visualised by in situ hybridisation. Recent digital PCR data indicate the mutant transcript is present in a 7x excess compared to the wild-type transcript. This transcript is tagged for degradation by a mechanism that is not understood, but which likely involves RNA modification. This project involves studies based on the unique cell lines from which the mutant RNA will be captured using complementary bead-pull down and analysed by a series of molecular, cellular and chemical methods. The sequencing part of the project will involve RNA-modified standards prepared in the School of Chemistry. In addition to the cell lines described above, the project will involve CRISPR Cas9 genome engineering to introduce an inducible promoter upstream of the DNA repeat expansion such that expression of the mutant RNA can be controlled by doxycycline induction. This will permit higher levels of the mutant transcript to be generated for capture and analysis. The repeat expansion RNA will be analysed using a series of techniques including super resolution microscopy, Nanopore sequencing, thin layer chromatography and mass spectrometry. The aim of the project is to study RNA modification at single nucleotide resolution to provide an insight to the molecular processes underpinning RNA metabolism.