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.
Abstract
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.
N6-methyladenosine or m6A modification to mRNAs is now recognised as a key regulator of gene expression and protein translation. The fate of m6A-modified mRNAs is decoded by m6A readers, mostly found ...in the cytoplasm, except for the nuclear-localised YTHDC1. While earlier studies have implicated YTHDC1–m6A functions in alternative splicing and mRNA export, recent literature has expanded its close association to the chromatin-associated, noncoding and regulatory RNAs to fine-tune transcription and gene expression in cells. Here, we summarise current progress in the study of YTHDC1 function in cells, highlighting its multiple modes of action in regulating gene expression, and propose the formation of YTHDC1 nuclear condensates as a general mechanism that underlies its diverse functions in the nucleus.
YTHDC1 interacts with m6A-modified RNAs to regulate multiple steps of RNA metabolism in the nucleus.YTHDC1 is widely associated with transcriptional activation (via enhancer RNA-mediated crossregulation with active epigenetic marks).YTHDC1 transcriptional repressive action is largely associated with transposable elements and long ncRNAs.The diversity in YTHDC1–m6A functions is linked to their ability to promote membraneless nuclear subcompartments, such as the nuclear speckles.
Monocytes play a crucial role in maintaining homeostasis and mediating a successful innate immune response. They also act as central players in diverse pathological conditions, thus making them an ...attractive therapeutic target. Within the bone marrow, monocytes arise from a committed precursor termed Common Monocyte Progenitor (cMoP). However, molecular mechanisms that regulate the differentiation of cMoP to various monocytic subsets remain unclear. Herein, we purified murine myeloid precursors for deep poly‐A‐enriched RNA sequencing to understand the role of alternative splicing in the development and differentiation of monocytes under homeostasis. Our analyses revealed intron retention to be the major alternative splicing mechanism involved in the monocyte differentiation cascade, especially in the differentiation of Ly6Chi monocytes to Ly6Clo monocytes. Furthermore, we found that the intron retention of key genes involved in the differentiation of murine Ly6Chi to Ly6Clo monocytes was also conserved in humans. Our data highlight the unique role of intron retention in the regulation of the monocytic differentiation pathway.
We identify intron retention as a major alternative splicing mechanism that fine‐tunes the expression of key genes in the monocytic differentiation pathway. Our analysis shows conservation of intron retention events involved in the differentiation of Ly6Chi monocytes to Ly6Clo monocytes in both mouse and human.
•Intron retention is a mechanism of gene expression control in eukaryotes.•Custom computational pipelines are essential for IR detection.•Phylogenetic analyses reveal conserved IR and functional ...consequences.•Subcellular fractionation helps determine the spatio-functional relationship of IR.•Improvements in mass spectrometry are critical to detect novel IR-derived peptides.
RNA sequencing has revealed a striking diversity in transcriptomic complexity, to which alternative splicing is a major contributor. Intron retention (IR) is a conserved form of alternative splicing that was originally overlooked in normal mammalian physiology and development, due mostly to difficulties in its detection. IR has recently been revealed as an independent mechanism of controlling and enhancing the complexity of gene expression. IR facilitates rapid responses to biological stimuli, is involved in disease pathogenesis, and can generate novel protein isoforms. Many challenges, however, remain in detecting and quantifying retained introns and in determining their effects on cellular phenotype. In this review, we provide an overview of these challenges, and highlight approaches that can be used to address them.
Human and animal studies support that consuming a high level of linoleic acid (LA, 18:2ω‐6), an essential fatty acid and key component of the human diet, increases the risk of colon cancer. However, ...results from human studies have been inconsistent, making it challenging to establish dietary recommendations for optimal LA intake. Given the importance of LA in the human diet, it is crucial to better understand the molecular mechanisms underlying its potential colon cancer‐promoting effects. Using LC‐MS/MS‐based targeted lipidomics, we find that the cytochrome P450 (CYP) monooxygenase pathway is a major pathway for LA metabolism in vivo. Furthermore, CYP monooxygenase is required for the colon cancer‐promoting effects of LA, since the LA‐rich diet fails to exacerbate colon cancer in CYP monooxygenase‐deficient mice. Finally, CYP monooxygenase mediates the pro‐cancer effects of LA by converting LA to epoxy octadecenoic acids (EpOMEs), which have potent effects on promoting colon tumorigenesis via gut microbiota‐dependent mechanisms. Overall, these results support that CYP monooxygenase‐mediated conversion of LA to EpOMEs plays a crucial role in the health effects of LA, establishing a unique mechanistic link between dietary fatty acid intake and cancer risk. These results could help in developing more effective dietary guidelines for optimal LA intake and identifying subpopulations that may be especially vulnerable to LA's negative effects.
CYP monooxygenase‐mediated conversion of LA to EpOMEs contributes to the colon cancer‐enhancing effects of dietary LA. After a high intake of dietary LA, LA is metabolized by CYP monooxygenases (largely CYP2C and CYP2J isoforms), which are upregulated in colon tumors, leading to the formation of EpOMEs. EpOMEs have potent effects to induce colon tumorigenesis via gut microbiota‐dependent mechanisms. In contrast, DiHOMEs, which are downstream metabolites of EpOMEs, do not have such effects. Overall, these results support that CYP monooxygenase‐mediated conversion of LA to EpOMEs plays a critical role in mediating the colon cancer‐enhancing effects of dietary LA.
Universal screening of endometrial carcinoma (EC) for mismatch repair deficiency (MMRd) and Lynch syndrome uses presence of MLH1 methylation to omit common sporadic cases from follow-up germline ...testing. However, this overlooks rare cases with high-risk constitutional MLH1 methylation (epimutation), a poorly-recognized mechanism that predisposes to Lynch-type cancers with MLH1 methylation. We aimed to determine the role and frequency of constitutional MLH1 methylation among EC cases with MMRd, MLH1-methylated tumors.
We screened blood for constitutional MLH1 methylation using pyrosequencing and real-time methylation-specific PCR in patients with MMRd, MLH1-methylated EC ascertained from (i) cancer clinics (n = 4, <60 years), and (ii) two population-based cohorts; “Columbus-area” (n = 68, all ages) and “Ohio Colorectal Cancer Prevention Initiative (OCCPI)” (n = 24, <60 years).
Constitutional MLH1 methylation was identified in three out of four patients diagnosed between 36 and 59 years from cancer clinics. Two had mono−/hemiallelic epimutation (∼50% alleles methylated). One with multiple primaries had low-level mosaicism in normal tissues and somatic “second-hits” affecting the unmethylated allele in all tumors, demonstrating causation. In the population-based cohorts, all 68 cases from the Columbus-area cohort were negative and low-level mosaic constitutional MLH1 methylation was identified in one patient aged 36 years out of 24 from the OCCPI cohort, representing one of six (∼17%) patients <50 years and one of 45 patients (∼2%) <60 years in the combined cohorts. EC was the first/dual-first cancer in three patients with underlying constitutional MLH1 methylation.
A correct diagnosis at first presentation of cancer is important as it will significantly alter clinical management. Screening for constitutional MLH1 methylation is warranted in patients with early-onset EC or synchronous/metachronous tumors (any age) displaying MLH1 methylation.
•High-risk constitutional MLH1 methylation underlies a significant proportion of early-onset EC with tumor MLH1 methylation.•EC with tumor MLH1 methylation is sometimes the ‘sentinel’ cancer in women with constitutional MLH1 methylation.•Low-level mosaic constitutional MLH1 methylation confers high-risk for MLH1-methylated cancers including EC.•Constitutional MLH1 methylation testing is warranted in cases with early-onset, or prior history of, MLH1-methylated cancer.
After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in ...cancer. Many solid and liquid tumours are associated with drastic increases in IR, and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue.
In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR.
Strikingly, we found that aberrantly decreasing IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA-binding protein expression and changes in tissue composition are among the causes of aberrantly decreasing IR in BrCa.
Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.
Intron retention (IR) is a form of alternative splicing that can affect mRNA levels through nonsense-mediated decay or by nuclear mRNA detention. A complex, dynamic IR pattern has been described in ...maturing mammalian granulocytes, but it is unknown whether IR occurs broadly in other hematopoietic lineages. We globally assessed IR in primary maturing mammalian erythroid and megakaryocyte (MK) lineages as well as their common progenitor cells (MEPs). Both lineages exhibit an extensive differential IR program involving hundreds of introns and genes with an overwhelming loss of IR in erythroid cells and MKs compared with MEPs. Moreover, complex IR patterns were seen throughout murine erythroid maturation. Similarly complex patterns were observed in human erythroid differentiation, but not involving the murine orthologous introns or genes. Despite the common origin of erythroid cells and MKs, and overlapping gene expression patterns, the MK IR program is entirely distinct from that of the erythroid lineage with regards to introns, genes, and affected gene ontologies. Importantly, our results suggest that IR serves to broadly regulate mRNA levels. These findings highlight the importance of this understudied form of alternative splicing in gene regulation and provide a useful resource for studies on gene expression in the MK and erythroid lineages.
•Dynamic intron retention programs exist in the murine megakaryocyte and erythroid and human erythroid lineages.•Intron retention inversely correlates with expression levels of a large set of transcripts.
Intron retention (IR) is a form of alternative splicing that has long been neglected in mammalian systems although it has been studied for decades in non-mammalian species such as plants, fungi, ...insects and viruses. It was generally assumed that mis-splicing, leading to the retention of introns, would have no physiological consequence other than reducing gene expression by nonsense-mediated decay. Relatively recent landmark discoveries have highlighted the pivotal role that IR serves in normal and disease-related human biology. Significant technical hurdles have been overcome, thereby enabling the robust detection and quantification of IR. Still, relatively little is known about the cis- and trans-acting modulators controlling this phenomenon. The fate of an intron to be, or not to be, retained in the mature transcript is the direct result of the influence exerted by numerous intrinsic and extrinsic factors at multiple levels of regulation. These factors have altered current biological paradigms and provided unexpected insights into the transcriptional landscape. In this review, we discuss the regulators of IR and methods to identify them. Our focus is primarily on mammals, however, we broaden the scope to non-mammalian organisms in which IR has been shown to be biologically relevant.
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