Identification of novel microproteins related to epitranscriptomic mechanisms
Publication Date
July 27, 2026
Creator
Stones, Joseph Harry
Abstract
In recent years, previously unknown complexities in the regulation of RNA and protein systems have been uncovered. One such regulatory mechanism of RNA is the chemical modification of nucleotides, for instance, the m6A modification. This common modification is governed by ‘effector’ proteins. It is added by ‘writers’, removed by ‘erasers’, and selectively interacts with ‘readers’. m6A has been linked to RNA metabolism, and the translation of alternative mRNA isoforms. Dysregulation of m6A has been associated with cellular dysfunction and various diseases, including neurological disorders and cancers.
At the protein level, it has recently been found that there is abundant translation of proteins smaller than 100 amino acids, termed ‘microproteins’. Microproteins have previously evaded detection due to size cutoffs in genome annotations and limitations in typical mass spectrometry experiments and software. However, ribosome profiling has emerged as a powerful technique for identifying microprotein translation in short regions of RNA, or ‘small open reading frames’ (sORFs). Many sORFs were previously thought to be noncoding, but it now appears that there are many unidentified microproteins which play important roles in various cellular processes and diseases.
There is currently sparse evidence in the literature, just one study, directly linking m6A to sORF translation. Due to this and m6A’s role in alternative translation, this thesis aimed to explore a more widespread relationship between the m6A modification, effector proteins, and sORF translation.
To accomplish these aims, I first analysed RNA-sequencing data from eleven human cell line studies where an m6A writer, reader, or eraser was repressed to identify differentially expressed transcripts compared to control conditions. Cellular processes commonly affected by effector protein silencing were identified, including the neuron-related terms ‘axon guidance’ and ‘calcium ion binding’, which were downregulated following writer silencing. Noncoding transcripts with unknown functions had their functionality predicted based on their clustering with genes of known functions. One group of noncoding RNAs clustered with glutamatergic synapse related transcripts that are known to be m6A modified, marking them as interesting targets for the identification of microproteins related to m6A and synaptic processes.
For microprotein discovery, ribosome profiling data from five human tissue or cell line studies were analysed to create a catalogue of over one million sORFs which showed evidence of translation across multiple samples. These were characterised further, to investigate: evolutionary conservation, translation initiation motif strength, predicted cellular localisation, and domain homology of the protein product. A subset of sORFs were found to have characteristics similar to canonical protein coding genes and their structures were predicted using AlphaFold, revealing short alpha helices as a potential common motif of microproteins.
The sORF catalogue and RNA-sequencing data were combined to identify sORFs within differentially expressed transcripts. Additionally, sORFs within effector protein transcripts, or transcripts from nearby genomic locations were identified. One predicted microprotein was encoded by the noncoding RBM33-DT gene, which neighbours the m6A reader gene, RBM33. I hypothesised and characterised an oligomerisation interaction between the microprotein and RBM33. Finally, small proteins were enriched from human neuroblastoma cells, and mass spectrometry was used to find proteomic evidence of microproteins. Some of the identified microproteins came from transcripts found to be differentially expressed following m6A effector protein repression, implying that previously unknown microproteins are indeed involved in m6A related processes. These novel microproteins may have vital roles in cellular processes and disease pathways that are not fully understood, future experiments will be required to elucidate their specific functions.
Item Type
ethesis
Thesis Type
PhD
Supervisors
University of Nottingham
University of Nottingham
University of Nottingham
Subjects (LC)
Associated Schools / Departments
UoN Repository URI
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