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

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.