Bao-Fa Sun

Nuclear m6A Reader YTHDC1 Regulates mRNA Splicing

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis

The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.

m6A modulates haematopoietic stem and progenitor cell specification

N6-methyladenosine (m6A) has been identified as the most abundant modification on eukaryote messenger RNA (mRNA). Although the rapid development of high-throughput sequencing technologies has enabled insight into the biological functions of m6A modification, the function of m6A during vertebrate embryogenesis remains poorly understood. Here we show that m6A determines cell fate during the endothelial-to-haematopoietic transition (EHT) to specify the earliest haematopoietic stem/progenitor cells (HSPCs) during zebrafish embryogenesis. m6A-specific methylated RNA immunoprecipitation combined with high-throughput sequencing (MeRIP–seq) and m6A individual-nucleotide-resolution cross-linking and immunoprecipitation with sequencing (miCLIP–seq) analyses reveal conserved features on zebrafish m6A methylome and preferential distribution of m6A peaks near the stop codon with a consensus RRACH motif. In mettl3-deficient embryos, levels of m6A are significantly decreased and emergence of HSPCs is blocked. Mechanistically, we identify that the delayed YTHDF2-mediated mRNA decay of the arterial endothelial genes notch1a and rhoca contributes to this deleterious effect. The continuous activation of Notch signalling in arterial endothelial cells of mettl3-deficient embryos blocks EHT, thereby repressing the generation of the earliest HSPCs. Furthermore, knockdown of Mettl3 in mice confers a similar phenotype. Collectively, our findings demonstrate the critical function of m6A modification in the fate determination of HSPCs during vertebrate embryogenesis.

5-methylcytosine promotes mRNA export — NSUN2 as the methyltransferase and ALYREF as an m 5 C reader

5-methylcytosine (m5C) is a post-transcriptional RNA modification identified in both stable and highly abundant tRNAs and rRNAs, and in mRNAs. However, its regulatory role in mRNA metabolism is still largely unknown. Here, we reveal that m5C modification is enriched in CG-rich regions and in regions immediately downstream of translation initiation sites and has conserved, tissue-specific and dynamic features across mammalian transcriptomes. Moreover, m5C formation in mRNAs is mainly catalyzed by the RNA methyltransferase NSUN2, and m5C is specifically recognized by the mRNA export adaptor ALYREF as shown by in vitro and in vivo studies. NSUN2 modulates ALYREFs nuclear-cytoplasmic shuttling, RNA-binding affinity and associated mRNA export. Dysregulation of ALYREF-mediated mRNA export upon NSUN2 depletion could be restored by reconstitution of wild-type but not methyltransferase-defective NSUN2. Our study provides comprehensive m5C profiles of mammalian transcriptomes and suggests an essential role for m5C modification in mRNA export and post-transcriptional regulation.

Smg6/Est1 licenses embryonic stem cell differentiation via nonsense-mediated mRNA decay

Nonsense‐mediated mRNA decay (NMD) is a post‐transcriptional mechanism that targets aberrant transcripts and regulates the cellular RNA reservoir. Genetic modulation in vertebrates suggests that NMD is critical for cellular and tissue homeostasis, although the underlying mechanism remains elusive. Here, we generate knockout mice lacking Smg6/Est1, a key nuclease in NMD and a telomerase cofactor. While the complete loss of Smg6 causes mouse lethality at the blastocyst stage, inducible deletion of Smg6 is compatible with embryonic stem cell (ESC) proliferation despite the absence of telomere maintenance and functional NMD. Differentiation of Smg6‐deficient ESCs is blocked due to sustained expression of pluripotency genes, normally repressed by NMD, and forced down‐regulation of one such target, c‐Myc, relieves the differentiation block. Smg6‐null embryonic fibroblasts are viable as well, but are refractory to cellular reprograming into induced pluripotent stem cells (iPSCs). Finally, depletion of all major NMD factors compromises ESC differentiation, thus identifying NMD as a licensing factor for the switch of cell identity in the process of stem cell differentiation and somatic cell reprograming.

FTO and Obesity: Mechanisms of Association

The Fat mass and obesity associated (FTO) gene is a newly identified genetic factor for obesity. However, the exact molecular mechanisms responsible for the effect of FTO on obesity remain largely unknown. Recent studies from genome-wide associated studies reveal that genetic variants in the FTO gene are associated not only with human adiposity and metabolic disorders, but also with cancer, a highly obesity-associated disease as well. Data from animal and cellular models further demonstrate that the perturbation of FTO enzymatic activity dysregulates genes related to energy metabolism, causing the malfunction of energy and adipose tissue homeostasis in mice. The most significant advance about FTO research is the recent discovery of FTO as the first N6-methyl-adenosine (m6A) RNA demethylase that catalyzes the m6A demethylation in α-ketoglutarate - and Fe2+-dependent manners. This finding provides the strong evidence that the dynamic and reversible chemical m6A modification on RNA may act as a novel epitranscriptomic marker. Furthermore, the FTO protein was observed to be partially localized onto nuclear speckles enriching mRNA processing factors, implying a potential role of FTO in regulating RNA processing. This review summarizes the recent progress about biological functions of FTO through disease-association studies as well as the data from in vitro and in vivo models, and highlights the biochemical features of FTO that might be linked to obesity.

Mettl3-mediated m 6 A regulates spermatogonial differentiation and meiosis initiation

METTL3 catalyzes the formation of N6-methyl-adenosine (m6A) which has important roles in regulating various biological processes. However, the in vivo function of Mettl3 remains largely unknown in mammals. Here we generated germ cell-specific Mettl3 knockout mice and demonstrated that Mettl3 was essential for male fertility and spermatogenesis. The ablation of Mettl3 in germ cells severely inhibited spermatogonial differentiation and blocked the initiation of meiosis. Transcriptome and m6A profiling analysis revealed that genes functioning in spermatogenesis had altered profiles of expression and alternative splicing. Our findings provide novel insights into the function and regulatory mechanisms of Mettl3-mediated m6A modification in spermatogenesis and reproduction in mammals.

METTL3-mediated m6A modification is required for cerebellar development

N6-methyladenosine (m6A) RNA methylation is the most abundant modification on mRNAs and plays important roles in various biological processes. The formation of m6A is catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. However, the in vivo functions of METTL3 and m6A modification in mammalian development remain unclear. Here, we show that specific inactivation of Mettl3 in mouse nervous system causes severe developmental defects in the brain. Mettl3 conditional knockout (cKO) mice manifest cerebellar hypoplasia caused by drastically enhanced apoptosis of newborn cerebellar granule cells (CGCs) in the external granular layer (EGL). METTL3 depletion–induced loss of m6A modification causes extended RNA half-lives and aberrant splicing events, consequently leading to dysregulation of transcriptome-wide gene expression and premature CGC death. Our findings reveal a critical role of METTL3-mediated m6A in regulating the development of mammalian cerebellum.

Circulating tumor DNA 5-hydroxymethylcytosine as a novel diagnostic biomarker for esophageal cancer

Dear Editor, Esophageal cancer is a serious malignancy with high rates of incidence and mortality. It ranked the eighth most common cancer and the sixth leading cause of cancer death worldwide. About 87% of esophageal cancers are esophageal squamous cell carcinomas (ESCCs), with the highest incidence found in SouthEastern and Central Asia. Notably, almost half of the global esophageal cancers occur in China. The 5-year survival rate of esophageal cancer patients is only 15 to 20%, partly due to late clinical presentation and lack of early diagnostic biomarkers. Therefore, exploring a highly sensitive and specific early diagnosis method is in urgent need. One of the early events that occur during carcinogenesis is epigenetic alterations, including aberrant DNA and histone modifications. Circulating cell-free DNA (cfDNA) in plasma has been shown to reflect the epigenetic features in cancer patients. 5-hydroxymethylcytosine (5hmC), the oxidative product of 5methylcytosine (5mC) catalyzed by ten-eleven translocation protein family, is not only a relatively stable intermediate of active DNA demethylation, but also regarded as a novel epigenetic hallmark of cancer. Two recent studies have revealed that 5hmC patterns in cfDNA provide tumor-associated signatures of several human cancers. Therefore, 5hmCs in cfDNA have potential to be promising biomarkers for minimally invasive diagnosis in esophageal cancer. Here, we utilized our recently established nano-hmC-Seal method to map the 5hmC profiles in cfDNA from a cohort of 150 newly diagnosed esophageal cancer patients and 177 healthy individuals (Fig. 1a; Supplementary information, Figure S1 and Table S1). In addition to ESCCs, there were nine adenocarcinoma, three small cell carcinoma, and one neuroendocrine carcinoma samples in our cohort, identified by hematoxylin and eosin staining (Fig. 1b). We first identified the 5hmC-enriched peaks and found that the peak numbers were more stable in control samples than esophageal samples (Supplementary information, Figure S2A). The heterogeneity of tumor samples may contribute to this result. Then we evaluated the distribution of 5hmC along the gene bodies in tumor and control groups. Compared to controls, tumor groups showed some increased 5hmC levels in gene bodies (Supplementary information, Figure S2B). Then, we identified differentially hydroxymethylated regions (DhMRs) and detected 5hmC-gain regions (9,047) and 5hmC-loss regions (10,460) in tumor groups by comparing tumor groups with control groups. 5hmC-gain regions, but not the 5hmC-loss regions, were particularly enriched in promoter and UTR regions. (Supplementary information, Figure S2C, D). Meanwhile, we found the enrichment of 5hmC-gain regions in short interspersed nuclear element, long tandem repeat, and satellite repeats. (Supplementary information, Figure S2D). All these results suggest that cfDNA 5hmC profiles of healthy individuals and esophageal cancer patients indeed display significant differences. To better understand the correlation between regulatory sequence codes and 5hmC changes, we performed de novo motif analysis in DhMRs. Most of the 5hmC peaks in 5hmC-gain regions were enriched in CEBP motif (CCAAT/enhancer-binding protein epsilon, p= 1e), which was highly related to transcriptional misregulation in cancer. In contrast, ARNT motif was observed in the 5hmC-loss regions. It was known that the heterodimer composed of ARNT and HIF1A acted as a transcriptional regulator of adaptive response to hypoxia. Thus, the enrichment of this heterodimer may be the result of hypoxia (Supplementary information, Figure S2E). To further explore the 5hmC signal changes between tumor and non-tumor samples, we then detected the differentially regulated 5hmC genes (i.e., genes with differential 5hmC levels) in esophageal cancer samples with DESeq2 package. The results showed that esophageal cancer could lead to both upregulated and downregulated 5hmC levels in genes compared to control individuals (up 1,344 and down 231). To further validate the classification effects of 5hmC signal for esophageal cancer and control samples, we clustered the genes with differentially regulated 5hmC levels by hierarchical clustering method and the results showed that the majority of cancer samples were distinct from non-cancer samples (Fig. 1c). However, both cancer and non-cancer samples in cluster 2 showed similar patterns. Then we carried out the PCA (principal component analysis) analysis for genes with differentially regulated 5hmC levels and found that esophageal cancer samples showed distinct signatures and could be readily separated from control samples (Fig. 1d). Meanwhile, the four categories obtained by the clustering analyses could also be separated from each other in PCA result (Supplementary information, Figure S3A). In contrast, the samples from different age range were not separated (Supplementary information, Figure S3B). The hierarchical clustering and PCA analysis based on top variance genes showed similar results (Supplementary information, Figure S3C, D). Next, to explore the function of differentially regulated 5hmC genes in esophageal cancer, we did the functional enrichment analysis for genes with upregulated and downregulated 5hmC levels in esophageal cancer, respectively (Fig. 1e, f). Our analyses showed cancer-related and metastasisrelated pathways such as Hippo signaling pathway, platelet homeostasis, PI3k–Akt signaling pathway, and MAPK signaling pathway were enriched. Together, these results indicate that the 5hmC signal within genes display obvious difference between the esophageal cancer and control and these differentially regulated 5hmC genes are enriched in pathways associated with cancer and metastasis. In addition, we further compared our data to

5-Hydroxymethylome in Circulating Cell-free DNA as A Potential Biomarker for Non-small-cell Lung Cancer

Non-small-cell lung cancer (NSCLC), the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymethylcytosine (5hmC) signatures in circulating cell-free DNA (cfDNA) that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminating tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here, we employed a sensitive and reliable method to map genome-wide 5hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identified six potential biomarkers from 66 patients and 67 healthy controls (mean decrease accuracy >3.2, P < 3.68E−19) using machine-learning-based tumor classifiers with high accuracy. Thus, the unique signature of 5hmC in tumor patient’s cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.