Investigation of m6A-regulated RNA-binding proteins

Abstract

N6-methyladenosine (m6A) is one of the most ubiquitous modifications of eukaryotic RNAs. The modifi- cation functions to control the normal processing, export, splicing, and metabolism of cellular RNAs. All of these processes are mediated by a group of m6A RNA-binding proteins known as ‘readers’ that bind modified RNA - directly or indirectly - and recruit other protein complexes to alter RNA fate. Canonical m6A readers include the YTH family of proteins which directly interact with the modification in specific sequence motifs via a deep hydrophobic cleft in their YTH-domain. Beyond the YTH proteins are sev- eral more generic RNA-binding proteins that moonlight as non-canonical m6A readers. The most well characterised of these proteins are the IGF2BP (IMP) family of proteins which have also been shown to directly interact with m6A, but with more modest affinities as m6A-binding is not necessarily their primary function. Over the years other proposed non-canonical m6A readers have been identified but in a piecemeal fashion. My thesis describes a project focused on the identification of cis- and trans-m6A RNA-binding proteins at a system-wide level. I harnessed recently described small molecule inhibitors for METTL3 (the primary m6A methyltrans- ferase) to perturb cellular m6A and study the effects on RNA-binding proteins in MOLM-13, an acute myeloid leukaemia (AML) cell line. This was coupled with the OOPS quantitative proteomics technique to directly measure in the cell which RNA-proteins had their binding levels significantly altered by the loss of the modification. With this data I looked for groups of proteins that behaved in a similar man- ner upon m6A depletion, regardless of any overall changes in total protein abundance. Following this I performed an orthogonal m6A RNA affinity purification assay to identify proteins that showed in vitro affinity for an m6A RNA oligo over its unmodified equivalent. Proteins highlighted in both assays showed reduced RNA-binding in the cell with modification loss and increased RNA-binding affinity, suggesting a putative function involving m6A RNA. Lastly, the proteomics experiments were followed up by a focused iiCLIP experiment, with and without m6A depletion, on a single candidate non-canonical m6A reader to characterise its m6A regulated binding sites. This investigation provides a broad overview of the m6A- regulated RBPome, which has not previously been investigated at a system-wide level. Additionally, I identify a number of novel putative m6A readers which are ripe for future investigation.