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RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m6A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m6A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m6A and 5 hmC reveals enrichment of m6A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m6A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m6A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m6A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) is a crucial mechanism for regulating gene expression and isoform diversity in eukaryotes. However, the analysis and visualization of AS events from RNA sequencing data remains challenging. Most tools require a certain level of computer literacy and the available means of visualizing AS events, such as coverage and sashimi plots, have limitations and can be misleading. To address these issues, we present SpliceWiz , an R package with an interactive Shiny interface that allows easy and efficient AS analysis and visualization at scale. A novel normalization algorithm is implemented to aggregate splicing levels within sample groups, thereby allowing group differences in splicing levels to be accurately visualized. The tool also offers downstream gene ontology enrichment analysis, highlighting ASEs belonging to functional pathways of interest. SpliceWiz is optimized for speed and efficiency and introduces a new file format for coverage data storage that is more efficient than BigWig. Alignment files are processed orders of magnitude faster than other R-based AS analysis tools and on par with command-line tools. Overall, SpliceWiz streamlines AS analysis, enabling reliable identification of functionally relevant AS events for further characterization. SpliceWiz is a Bioconductor package and is also available on GitHub (https://github.com/alexchwong/SpliceWiz). [ABSTRACT FROM AUTHOR]

Background: N7-methylguanosine (m 7 G) modification is one of the most prevalent RNA modifications in humans. Dysregulated m 7 G modifications caused by aberrant expression of m 7 G writers contribute to cancer progression and result in worse patient survival in several human cancers. However, studies that systematically assess the frequency and clinical relevance of aberrant m 7 G writer expression in a pan-cancer cohort remain to be performed., Aims: This study aims to systematically investigate the molecular alteration and clinical relevance of m 7 G methyltransferase in human cancers., Methods: We analysed genome, transcriptome and clinical data from the Cancer Genome Atlas Research Network spanning 33 types of human cancers for aberrant changes in genes encoding m 7 G writers., Result: We demonstrate that m 7 G writers are dysregulated in human cancers and are associated predominantly with poorer survival. By dividing patients into those with high and low m 7 G scores, we show that a lower m 7 G score is generally associated with immune infiltration and better response to immunotherapy., Conclusion: Our analyses indicate the genetic alterations, expression patterns and clinical relevance of m 7 G writers across various cancers. This study provides insights into the potential utility of m 7 G writer expression as a cancer biomarker and proposes the possibility of targeting m 7 G writers for cancer therapy., (© 2024 The Author(s). Cancer Reports published by Wiley Periodicals LLC.)

RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. While m 6 A has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled m 6 A and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of m 6 A and 5 hmC reveals enrichment of m 6 A and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that m 6 A and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of m 6 A and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of m 6 A. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity., (© 2024. The Author(s).)