Bellows, Eleanor
(2020)
Biological functions of cap adjacent RNA methylation in brain.
PhD thesis, University of Nottingham.
Abstract
In eukaryotes, mRNA transcripts that are transcribed by RNA polymerase II (RNA pol II) and then subsequently co-transcriptionally modified. One of the co-transcriptional modifications is the addition of the 7-methyl guanine cap to the 5′ end of the RNA. If no further modifications are added to the first two nucleotides, this cap structure is referred to as cap0, and is found in plants and yeast. Additional modifications take place in higher eukaryotes. For example the ribose sugar of the first nucleotide can be methylated at the 2′-O position to give cap1. If this cap1 methylation occurs on an adenosine, the nitrogenous base can be further methylated generating N6,2′-O-dimethyladenosine (m6Am). The function of the m6Am modification is currently unknown. We propose that the m6Am modification may regulate translational processes which underlie neuronal and synaptic function. To understand this, transcripts that have the modification, proteins that bind the modification and evolutionary profile of m6Am will be investigated by preforming Thin Layer Chromatography (TLCs), protein pull downs using RNA oligonucleotides and RNA sequencing and gene orthologue analysis.
Within the evolutionary tree, m6Am was found in vertebrates and preliminary evidence suggests its presence in the marine algae E. siliculosus. The organisms which contain m6Am methylation all had CMTR1, (the Am methylase), PCIF1, (the m6Am methylase) and FTO, (the m6Am demethylase). However, the invertebrate species where m6Am was absent, also contained PCIF1 and CMTR1 enzymes.
In this study, proteins binding to the Am and m6Am modification were found to be different indicating that Am has a different role than m6Am. Am-binding proteins were found to be associated with RNA processing such as splicing, removal of RNA secondary structures and translation. In contrast m6Am binding proteins were shown to be associated with microtubules and extracellular vesicles suggesting a function in RNA transport throughout the cell and between cells.
Previously DCP2 was reported to not decap transcripts that started with m6Am, it was hypothesised that if this could be confirmed it could be used in a novel enzymatic enrichment sequencing method for m6Am. Contrary to this published data DCP2 was found to preferentially decap Am and m6Am transcripts. This information combined with the activity of other known decapping enzymes highlights a potential mechanism of regulating different sub-populations of RNA denoted by the cap structure.
Putative m6Am methylated transcripts were identified using a bioinformatic approach from m6A sequencing of human foetal brain, and parahippocampus brain- grey matter and white matter. Cell type specific transcripts were indicated to be methylated suggesting that m6Am may increase translation or stability of these transcripts. An increase in global Am and m6Am abundance in Parkinson’s disease and Lewy body dementia-affected hippocampus tissue specimens was observed. Further research is needed to provide insight into which specific transcripts show changes in Am and m6Am methylation as the diseases progress.
Taken together, these results suggest that the function of the m6Am modification is context dependant. At the molecular level, m6Am may instigate the same effect across all cells but it is m6Am binding proteins and the individual transcripts that are methylated that provide the unique cell specific regulation of mRNA fate. Observations made in this thesis provide important questions for further research into the role of m6Am in brain function and neurodegenerative diseases.
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