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RNA modifications have emerged as central regulators of gene expression programs. Amongst RNA modifications are N6-methyladenosine (m 6 A) and RNA 5-hydroxymethylcytosine (5hmC). While m 6 A is established as a versatile regulator of RNA metabolism, the functions of RNA 5hmC are unclear. Despite some evidence linking RNA modifications to immunity, their implications in gene expression control in macrophage development and functions remain unclear. Here we present a multi-omics dataset capturing different layers of the gene expression programs driving macrophage differentiation and polarisation. We obtained mRNA-Seq, m 6 A-IP-Seq, 5hmC-IP-Seq, Polyribo-Seq and LC-MS/MS data from monocytes and resting-, pro- and anti-inflammatory-like macrophages. We present technical validation showing high quality and correlation between samples for all datasets, and evidence of biological consistency of modelled macrophages at the transcriptomic, epitranscriptomic, translational and proteomic levels. This multi-omics dataset provides a resource for the study of RNA m 6 A and 5hmC in the context of macrophage biology and spans the gene expression process from transcripts to proteins., (© 2024. The Author(s).)

The aged brain is associated with an inevitable decline in cognitive function and increased vulnerability to neurodegenerative disorders. Multiple molecular hallmarks have been associated with the aging nervous system through transcriptomics and proteomic studies. Recently, epitranscriptomic analysis has highlighted the role of RNA chemical modification in various biological processes. In particular, N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNAs, has been functionally linked to multiple aspects of RNA metabolism with the roles of m6A in processes such as learning and memory, leading to our current investigation of how the m6A-transcriptomic landscape is shaped during aging. Using the inbred C57BL/6 line, we compared the m6A-transcriptomic profiles from the hippocampi of young (3-month-old) and aged (20-month-old) mice. Methylated RNA immunoprecipitation (MeRIP)-sequencing analysis revealed hyper- and hypomethylation in 426 and 102 genes, respectively, in the aged hippocampus (fold change >1.5, false discovery rate <0.05). By correlating the methylation changes to their steady-state transcript levels in the RNA-Seq data, we found a significant concordance between m6A and transcript levels in both directions. Notably, the myelin regulator gene Gpr17 was downregulated in the aged hippocampus concomitant with reduced m6A levels in its 3'UTR. Using reporter constructs and mutagenesis analysis, we demonstrated that the putative m6A sites in the 3'UTR of Gpr17 are important for mRNA translation but not for regulating transcript stability. Overall, the positive correlation between m6A and the transcript expression levels indicates a co-transcriptional regulation of m6A with gene expression changes that occur in the aged mouse hippocampus., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences immediately downstream of transcription start sites. Here, using cytoplasmic and nuclear small RNA high-throughput sequencing datasets, we report the identification of a second class of nuclear-specific approximately 17- to 18-nucleotide small RNAs whose 3' ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed genes and show evidence of developmental stage- and region-specific expression. We also show that tiRNAs are localized to the nucleus, are enriched at chromatin marks associated with transcription initiation and possess a 3'-nucleotide bias. Additionally, we find that microRNA-offset RNAs (moRNAs), the miR-15/16 cluster previously linked to oncosuppression and most small nucleolar RNA (snoRNA)-derived small RNAs (sdRNAs) are enriched in the nucleus, whereas most miRNAs and two H/ACA sdRNAs are cytoplasmically enriched. We propose that nuclear-localized tiny RNAs are involved in the epigenetic regulation of gene expression.

Simple Summary: This study provides new insights into the changing transcriptomic and epigenomic landscapes in chronic myeloid leukaemia (CML) patients who are receiving tyrosine kinase inhibitor (TKI) therapy (often life-long). Alternative splicing, vital for cellular homeostasis, is dysregulated in human cancers. Remarkably, we found abnormal splicing patterns despite molecular remission in peripheral blood cells of chronic-phase CML patients. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Vast transcriptomics and epigenomics changes are characteristic of human cancers, including leukaemia. At remission, we assume that these changes normalise so that omics-profiles resemble those of healthy individuals. However, an in-depth transcriptomic and epigenomic analysis of cancer remission has not been undertaken. A striking exemplar of targeted remission induction occurs in chronic myeloid leukaemia (CML) following tyrosine kinase inhibitor (TKI) therapy. Using RNA sequencing and whole-genome bisulfite sequencing, we profiled samples from chronic-phase CML patients at diagnosis and remission and compared these to healthy donors. Remarkably, our analyses revealed that abnormal splicing distinguishes remission samples from normal controls. This phenomenon is independent of the TKI drug used and in striking contrast to the normalisation of gene expression and DNA methylation patterns. Most remarkable are the high intron retention (IR) levels that even exceed those observed in the diagnosis samples. Increased IR affects cell cycle regulators at diagnosis and splicing regulators at remission. We show that aberrant splicing in CML is associated with reduced expression of specific splicing factors, histone modifications and reduced DNA methylation. Our results provide novel insights into the changing transcriptomic and epigenomic landscapes of CML patients during remission. The conceptually unanticipated observation of widespread aberrant alternative splicing after remission induction warrants further exploration. These results have broad implications for studying CML relapse and treating minimal residual disease. [ABSTRACT FROM AUTHOR]