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As an aberrant base in DNA, uracil is generated by either deoxyuridine (dU) misincorporation or cytosine deamination, and involved in multiple physiological and pathological processes. Genome-wide profiles of uracil are important for study of these processes. Current methods for whole-genome mapping of uracil all rely on uracil-DNA N-glycosylase (UNG) and are limited in resolution, specificity, and/or sensitivity. Here, we developed a UdgX cross-linking and polymerase stalling sequencing ("Ucaps-seq") method to detect dU at single-nucleotide resolution. First, the specificity of Ucaps-seq was confirmed on synthetic DNA. Then the effectiveness of the approach was verified on two genomes from different sources. Ucaps-seq not only identified the enrichment of dU at dT sites in pemetrexed-treated cancer cells with globally elevated uracil but also detected dU at dC sites within the "WRC" motif in activated B cells which have increased dU in specific regions. Finally, Ucaps-seq was utilized to detect dU introduced by the cytosine base editor (nCas9-APOBEC) and identified a novel off-target site in cellular context. In conclusion, Ucaps-seq is a powerful tool with many potential applications, especially in evaluation of base editing fidelity.

The mechanistic target of rapamycin complex 1 (mTORC1) acts as a central regulator of metabolic pathways that drive cellular growth. Abnormal activation of mTORC1 occurs at high frequency in human and mouse hepatocellular carcinoma (HCC). DEP domain-containing protein 5 (DEPDC5), a component of GATOR1 complex, is a repressor of amino acid-sensing branch of the mTORC1 pathway. In the current study, we found that persistent activation of hepatic mTORC1 signaling caused by Depdc5 ablation was sufficient to induce a pathological program of liver damage, inflammation and fibrosis that triggers spontaneous HCC development. Take advantage of the combinatory treatment with a single dose of diethylnitrosamine (DEN) and chronic feeding with high-fat diet (HFD), we demonstrated that hepatic depdc5 deletion did not aggravate DEN&HFD induced liver tumorigenesis, probably due to its protective effects on diet-induced liver steatosis. In addition, we further showed that chronic rapamycin treatment did not have any apparent tumor-suppressing effects on DEN&HFD treated control mice, whereas it dramatically reduced the tumor burden in mice with hepatic Depdc5 ablation. This study provides the novel in vivo evidence for Depdc5 deletion mediated mTORC1 hyperactivation in liver tumorigenesis caused by aging or DEN&HFD treatment. Moreover, our findings also propose that pharmacological inhibition of mTORC1 signaling maybe a promising strategy to treat HCC patients with mutations in DEPDC5 gene.

N 6‐methyladenosine (m6A) is the most prevalent modification in mRNA and engages in multiple biological processes. Previous studies indicated that m6A methyltransferase METTL3 (‘writer’) and demethylase FTO (‘eraser’) play critical roles in heart‐related disease. However, in the heart, the function of m6A ‘reader’, such as YTH (YT521‐B homology) domain‐containing proteins remains unclear. Here, we report that the defect in YTHDC1 but not other YTH family members contributes to dilated cardiomyopathy (DCM) in mice. Cardiac‐specific conditional Ythdc1 knockout led to obvious left ventricular chamber enlargement and severe systolic dysfunction. YTHDC1 deficiency also resulted in the decrease of cardiomyocyte contractility and disordered sarcomere arrangement. By means of integrating multiple high‐throughput sequence technologies, including m6A‐MeRIP, RIP‐seq and mRNA‐seq, we identified 42 transcripts as potential downstream targets of YTHDC1. Amongst them, we found that Titin mRNA was decorated with m6A modification and depletion of YTHDC1 resulted in aberrant splicing of Titin. Our study suggests that Ythdc1 plays crucial role in regulating the normal contractile function and the development of DCM. These findings clarify the essential role of m6A reader in cardiac biofunction and provide a novel potential target for the treatment of DCM.

The mammalian target of rapamycin complex 1 (mTORC1) is a crucial regulator of adipogenesis and systemic energy metabolism. Its dysregulation leads to a diversity of metabolic diseases, including obesity and type 2 diabetes. DEP-domain containing 5 (DEPDC5) is a critical component of GATOR1 complex that functions as a key inhibitor of mTORC1. So far, its function in adipose tissue remains largely unknown. Herein we evaluated how persistent mTORC1 activation in adipocyte via Depdc5 knockout modulates adiposity in vivo. Our data indicated that adipocyte-specific knockout of Depdc5 in aged mice led to reduced visceral fat, aggravated insulin resistance and enhanced adipose tissue inflammation. Moreover, we found that Depdc5 ablation resulted in upregulation of adipose triglyceride lipase (ATGL) in adipocytes and elevated levels of serum free fatty acids (FFAs). Intriguingly, rapamycin treatment did not reverse insulin resistance but alleviated adipose tissue inflammation caused by Depdc5 deletion. Taken together, our findings revealed that mTORC1 activation caused by Depdc5 deletion promotes lipolysis process and further exacerbates insulin resistance and adipose tissue inflammation in mice.

The asymmetric hydroboration of alkenes has proven to be among the most powerful methods for the synthesis of chiral boron compounds. This protocol is well suitable for activated alkenes such as vinylarenes and alkenes bearing directing groups. However, the catalytic enantioselective hydroboration of O-substituted alkenes has remained unprecedented. Here we report a Rh-catalyzed enantioselective hydroboration of silyl enol ethers (SEEs) that utilizes two new chiral phosphine ligands we developed. This approach features mild reaction conditions and a broad substrate scope as well as excellent functional group tolerance, and enables highly efficient preparation of synthetically valuable chiral borylethers.

Dilated cardiomyopathy, a type of heart muscle disease defined by the presence of left ventricular dilatation and contractile dysfunction, is an important cause of sudden cardiac death and heart failure. O-GlcNAcylation is an important post-translational modification of proteins by the addition of O-GlcNAc moieties at serine or threonine residues. Several studies have shown that proper control of O-GlcNAcylation is required for maintaining physiological function of heart by using Ogt (O-GlcNAc transferase) cardiomyocyte-specific knockout mouse models. In this study, we generated a new mouse model (αSMA-Ogt KO) in which Ogt was deleted in both cardiomyocytes and smooth muscle cells by crossing Ogt floxed mice with αSMA-Cre mice. αSMA-Cre-mediated Ogt deletion in mice led to severe postnatal lethality; the survived mice were smaller than control mice, had dilated hearts, and showed observable signs of heart failure. Moreover, the αSMA-Ogt KO heart had more apoptotic cells and fibrosis. The arteries of αSMA-Ogt KO mice exhibited significantly reduced expression of contractile genes and a trend towards arterial stiffness. In conclusion, our data emphasize the importance of OGT in maintaining normal heart function and reveal a novel role of OGT in regulating arterial contractility.

Activation of the heart normally begins in the sinoatrial node (SAN). Electrical impulses spontaneously released by SAN pacemaker cells (SANPCs) trigger the contraction of the heart. However, the cellular nature of SANPCs remains controversial. Here, we report that SANPCs exhibit glutamatergic neuron-like properties. By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse, we found that SANPCs co-clustered with cortical neurons. Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system, expressing genes encoding glutamate synthesis pathway (Gls), ionotropic and metabotropic glutamate receptors (Grina, Gria3, Grm1 and Grm5), and glutamate transporters (Slc17a7). SANPCs highly expressed cell markers of glutamatergic neurons (Snap25 and Slc17a7), whereas Gad1, a marker of GABAergic neurons, was negative. Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca2+ transients frequency in single SANPC. Collectively, our work suggests that SANPCs share dominant biological properties with glutamatergic neurons, and the glutamatergic neurotransmitter system may act as an intrinsic regulation module of heart rhythm, which provides a potential intervention target for pacemaker cell-associated arrhythmias.

METTL3 (methyltransferase-like 3) mediates the N6-methyladenosine (m6A) methylation of mRNA, which affects the stability of mRNA and its translation into protein1. METTL3 also binds chromatin2,3,4, but the role of METTL3 and m6A methylation in chromatin is not fully understood. Here we show that METTL3 regulates mouse embryonic stem-cell heterochromatin, the integrity of which is critical for silencing retroviral elements and for mammalian development5. METTL3 predominantly localizes to the intracisternal A particle (IAP)-type family of endogenous retroviruses. Knockout of Mettl3 impairs the deposition of multiple heterochromatin marks onto METTL3-targeted IAPs, and upregulates IAP transcription, suggesting that METTL3 is important for the integrity of IAP heterochromatin. We provide further evidence that RNA transcripts derived from METTL3-bound IAPs are associated with chromatin and are m6A-methylated. These m6A-marked transcripts are bound by the m6A reader YTHDC1, which interacts with METTL3 and in turn promotes the association of METTL3 with chromatin. METTL3 also interacts physically with the histone 3 lysine 9 (H3K9) tri-methyltransferase SETDB1 and its cofactor TRIM28, and is important for their localization to IAPs. Our findings demonstrate that METTL3-catalysed m6A modification of RNA is important for the integrity of IAP heterochromatin in mouse embryonic stem cells, revealing a mechanism of heterochromatin regulation in mammals.

Rationale: The stress response of heart rate, which is determined by the plasticity of the sinoatrial node (SAN), is essential for cardiac function and survival in mammals. As an RNA-binding protein, CIRP (cold-inducible RNA-binding protein) can act as a stress regulator. Previously, we have documented that CIRP regulates cardiac electrophysiology at posttranscriptional level, suggesting its role in SAN plasticity, especially upon stress conditions. Objective: Our aim was to clarify the role of CIRP in SAN plasticity and heart rate regulation under stress conditions. Methods and Results: Telemetric ECG monitoring demonstrated an excessive acceleration of heart rate under isoprenaline stimulation in conscious CIRP-KO (knockout) rats. Patch-clamp analysis and confocal microscopic Ca 2+ imaging of isolated SAN cells demonstrated that isoprenaline stimulation induced a faster spontaneous firing rate in CIRP-KO SAN cells than that in WT (wild type) SAN cells. A higher concentration of cAMP—the key mediator of pacemaker activity—was detected in CIRP-KO SAN tissues than in WT SAN tissues. RNA sequencing and quantitative real-time polymerase chain reaction analyses of single cells revealed that the 4B and 4D subtypes of PDE (phosphodiesterase), which controls cAMP degradation, were significantly decreased in CIRP-KO SAN cells. A PDE4 inhibitor (rolipram) abolished the difference in beating rate resulting from CIRP deficiency. The mechanistic study showed that CIRP stabilized the mRNA of Pde4b and Pde4d by direct mRNA binding, thereby regulating the protein expression of PDE4B and PDE4D at posttranscriptional level. Conclusions: CIRP acts as an mRNA stabilizer of specific PDEs to control the cAMP concentration in SAN, maintaining the appropriate heart rate stress response.

Objectives Accumulating evidences show that the regulatory network of m6A modification is essential for mammalian spermatogenesis. However, as an m6A reader, the roles of YTHDF2 remain enigmatic due to the lack of a proper model. Here, we employed the germ cell conditional knockout mouse model and explored the function of YTHDF2 in spermatogenesis. Materials and methods Ythdf2 germ cell conditional knockout mice were obtained by crossing Ythdf2‐floxed mice with Vasa‐Cre and Stra8‐Cre mice. Haematoxylin and eosin (HE) staining, immunofluorescent staining and Western blotting were used for phenotyping. CASA, IVF and ICSI were applied for sperm function analysis. RNA‐seq, YTHDF2‐RIP‐seq and quantitative real‐time PCR were used to explore transcriptome changes and molecular mechanism analysis. Results Our results showed that YTHDF2 was highly expressed in spermatogenic cells. The germ cell conditional knockout males were sterile, and their sperm displayed malformation, impaired motility, and lost fertilization ability. During differentiated spermatogonia transiting to pachytene spermatocyte, most m6A‐modified YTHDF2 targets that were degraded in control germ cells persisted in pachytene spermatocytes of Ythdf2‐vKO mice. These delayed mRNAs were mainly enriched in pathways related to the regulation of transcription, and disturbed the transcriptome of round spermatid and elongated spermatid subsequently. Conclusion Our data demonstrate that YTHDF2 facilitates the timely turnover of phase‐specific transcripts to ensure the proper progression of spermatogenesis, which highlights a critical role of YTHDF2 in spermatogenesis., YTHDF2 accelerates the degradation of m6A‐modified RNA during the early stages of spermatogenesis. The timely clearance of phase‐specific transcripts mediated by YTHDF2 ensures the formation of functional sperm.

Dynamic regulation of intestinal epithelial cell (IEC) differentiation is crucial for both homeostasis and the response to helminth infection. SIRT6 belongs to the NAD+-dependent deacetylases and has established diverse roles in aging, metabolism and disease. Here, we report that IEC Sirt6 deletion leads to impaired tuft cell development and type 2 immunity in response to helminth infection, thereby resulting in compromised worm expulsion. Conversely, after helminth infection, IEC SIRT6 transgenic mice exhibit enhanced epithelial remodeling process and more efficient worm clearance. Mechanistically, Sirt6 ablation causes elevated Socs3 expression, and subsequently attenuated tyrosine 641 phosphorylation of STAT6 in IECs. Notably, intestinal epithelial overexpression of constitutively activated STAT6 (STAT6vt) in mice is sufficient to induce the expansion of tuft and goblet cell linage. Furthermore, epithelial STAT6vt overexpression remarkedly reverses the defects in intestinal epithelial remodeling caused by Sirt6 ablation. Our results reveal a novel function of SIRT6 in regulating intestinal epithelial remodeling and mucosal type 2 immunity in response to helminth infection.

Host resistance to Mycobacterium tuberculosis infection requires the activities of multiple leukocyte subsets, yet the roles of the different innate effector cells during tuberculosis are incompletely understood. Here we uncover an unexpected association between eosinophils and Mtb infection. In humans, eosinophils are decreased in the blood but enriched in resected human tuberculosis lung lesions and autopsy granulomas. Influx of eosinophils is also evident in infected zebrafish, mice, and nonhuman primate granulomas, where they are functionally activated and degranulate. Importantly, employing complementary genetic models of eosinophil deficiency, we demonstrate that, in mice, eosinophils are required for optimal pulmonary bacterial control and host survival after Mtb infection. Collectively, our findings uncover an unexpected recruitment of eosinophils to the infected lung tissue and a protective role for these cells in the control of Mtb infection in mice.

The well-established functions of UHRF1 converge to DNA biological processes, as exemplified by DNA methylation maintenance and DNA damage repair during cell cycles. However, the potential effect of UHRF1 on RNA metabolism is largely unexplored. Here, we revealed that UHRF1 serves as a novel alternative RNA splicing regulator. The protein interactome of UHRF1 identified various splicing factors. Among them, SF3B3 could interact with UHRF1 directly and participate in UHRF1-regulated alternative splicing events. Furthermore, we interrogated the RNA interactome of UHRF1, and surprisingly, we identified U snRNAs, the canonical spliceosome components, in the purified UHRF1 complex. Unexpectedly, we found H3R2 methylation status determines the binding preference of U snRNAs, especially U2 snRNAs. The involvement of U snRNAs in UHRF1-containing complex and their binding preference to specific chromatin configuration imply a finely orchestrated mechanism at play. Our results provided the resources and pinpointed the molecular basis of UHRF1-mediated alternative RNA splicing, which will help us better our understanding of the physiological and pathological roles of UHRF1 in disease development.

Metabolic activity in pulmonary lesion is associated with disease severity and relapse risk in tuberculosis. However, the nature of the metabolic activity associated with tuberculosis in humans remains unclear. Previous works indicate that tuberculosis bears resemblance transcriptionally with systemic lupus erythematosus in peripheral blood, except that the plasma cell component was absent in tuberculosis. Here we reported that the missing transcriptional component was present within the metabolic active tissues in the lung of patients with sputum culture-negative tuberculosis, within which increased levels of circulating immune complexes and anti-dsDNA antibodies were found relative to nearby non-metabolic active tissues. Histological examination revealed specific vascular deposition of immune complexes, neutrophil extracellular traps, and vascular necrosis in the metabolic-active tissue. Thus, tuberculosis-initiated metabolic activity was associated with hyperactive antibody responses and vascular pathology, and shared features with systemic lupus erythematosus and other autoimmune diseases. We discussed these observations in the context of earlier literatures demonstrating that similar effects could be induced in humans and animal models by complete freund’s adjuvant, the most potent antibody response inducer ever reported. Our small case series, if verified in a larger size study, might help inform host-directed therapies to alleviate disease progression and augment treatment efficacy.IMPORTANCEIn patients with pulmonary tuberculosis, lung tissues were destroyed by a hyperactive inflammatory response towardsM. tuberculosis. The mechanisms underlying the inflammatory response are still poorly understood. Using 18F-FDG avidity as a surrogate marker of inflammation, we have identified that hyper-inflamed tissues possessed features associated with systemic lupus erythematosus: gene expression signatures of plasma cell and immunoglobulins and increased levels of anti-dsDNA antibodies, immune deposits, and vasculopathy. This observation might suggest an explanation to why patients with tuberculosis share more gene expression signatures with autoimmune diseases than infectious diseases and why they are more likely to develop autoimmune diseases. Defining the inflammatory responses at the lesion could help inform host-directed therapies to intervene disease progression or even accelerate cure.

RNA modifications represent a novel layer of regulation of gene expression. Functional experiments revealed that N(6)‐methyladenosine (m(6)A) on messenger RNA (mRNA) plays critical roles in cell fate determination and development. m(6)A mark also resides in the decoding center of 18S ribosomal RNA (rRNA); however, the biological function of m(6)A on 18S rRNA is still poorly understood. Here, we report that methyltransferase‐like 5 (METTL5) methylates 18S rRNA both in vivo and in vitro, which is consistent with previous reports. Deletion of Mettl5 causes a dramatic differentiation defect in mouse embryonic stem cells (mESCs). Mechanistically, the m(6)A deposited by METTL5 is involved in regulating the efficient translation of F‐box and WD repeat domain‐containing 7 (FBXW7), a key regulator of cell differentiation. Deficiency of METTL5 reduces FBXW7 levels and leads to the accumulation of its substrate c‐MYC, thereby delaying the onset of mESC differentiation. Our study uncovers an important role of METTL5‐mediated 18S m(6)A in mESC differentiation through translation regulation and provides new insight into the functional significance of rRNA m(6)A.

N6 methylation at adenosine 1832 (m

N6-methyladenosine (m6A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

Anthracyclines including doxorubicin (DOX) are still the most widely used and efficacious antitumor drugs, although their cardiotoxicity is a significant cause of heart failure. Despite considerable efforts being made to minimize anthracycline-induced cardiac adverse effects, little progress has been achieved. In this study, we aimed to explore the role and underlying mechanism of SNX17 in DOX-induced cardiotoxicity. We found that SNX17 was downregulated in cardiomyocytes treated with DOX both in vitro and in vivo. DOX treatment combined with SNX17 interference worsened the damage to neonatal rat ventricular myocytes (NRVMs). Furthermore, the rats with SNX17 deficiency manifested increased susceptibility to DOX-induced cardiotoxicity (myocardial damage and fibrosis, impaired contractility and cardiac death). Mechanistic investigation revealed that SNX17 interacted with leiomodin-2 (LMOD2), a key regulator of the thin filament length in muscles, via its C-TERM domain and SNX17 deficiency exacerbated DOX-induced cardiac systolic dysfunction by promoting aberrant LMOD2 degradation through lysosomal pathway. In conclusion, these findings highlight that SNX17 plays a protective role in DOX-induced cardiotoxicity, which provides an attractive target for the prevention and treatment of anthracycline induced cardiotoxicity.

N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as a new layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m6A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the nuclear processing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6A at its consensus motif. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance. Ythdc2 knockout mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those of littermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and accordingly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m6A-binding protein that plays critical roles during spermatogenesis.

As the most abundant modification on mRNA in mammal, N6-Methyladenosine (m6A) has been demonstrated to play important roles in various biological processes including mRNA splicing, translation and degradation. m6A reader proteins have been shown to play central roles in these processes. One of the m6A readers, YTHDF2 is localized to the P granules, which are liquid-like droplets where RNA degradation occurs. How YTHDF2 is localized to P granules is unknown. Here we provide evidence that YTHDF2 forms liquid droplets and phase separate, mediated by its low complexity (LC) domains. Interestingly, the ability to phase separate is robustly stimulated by m6A RNAs in vitro. In vivo, YTHDF2 phase separation may in fact be dependent on m6A RNA and YTHDF2 binding to m6A RNA, since a YTHDF2 m6A-binding defective mutant or a wildtype YTHDF2 assayed in cells lacking m6A RNAs, both fail to phase separate. The ability of phase separate is not limited to YTHDF2; we find other members of the YTH-domain m6A readers can also undergo phase separation. Our findings suggest that m6A RNA induced phase separation of m6A readers may play an important role in their distributions to different phase-separated compartments in cells.

N(6)-Methyladenosine (m(6)A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceo-somal RNAs (snRNA) in humans. Although mRNA m(6)A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m(6)A methyltransferases for rRNAs and the function of rRNA m(6)A modifications are unknown. Here we report a new m(6)A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m(6)A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m(6)A modification in translation and in tumor biology.

RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.

International audience; N6-Methyladenosine (m6A) RNA modification is present in messenger RNAs (mRNA), ribosomal RNAs (rRNA), and spliceosomal RNAs (snRNA) in humans. Although mRNA m6A modifications have been extensively studied and shown to play critical roles in many cellular processes, the identity of m6A methyltransferases for rRNAs and the function of rRNA m6A modifications are unknown. Here we report a new m6A methyltransferase, ZCCHC4, which primarily methylates human 28S rRNA and also interacts with a subset of mRNAs. ZCCHC4 knockout eliminates m6A4220 modification in 28S rRNA, reduces global translation, and inhibits cell proliferation. We also find that ZCCHC4 protein is overexpressed in hepatocellular carcinoma tumors, and ZCCHC4 knockout significantly reduces tumor size in a xenograft mouse model. Our results highlight the functional significance of an rRNA m6A modification in translation and in tumor biology.

N6 methylation at adenosine 1832 (m6A1832) of mammalian 18S rRNA, occupying a critical position within the decoding center, is modified by a conserved methyltransferase, METTL5. Here, we find that METTL5 shows strong substrate preference toward the 18S A1832 motif but not the other reported m6A motifs. Comparison with a yeast ribosome structural model unmodified at this site indicates that the modification may facilitate mRNA binding by inducing conformation changes in the mammalian ribosomal decoding center. METTL5 promotes p70-S6K activation and proper translation initiation, and the loss of METTL5 significantly reduces the abundance of polysome. METTL5 expression is elevated in breast cancer patient samples and is required for growth of several breast cancer cell lines. We further find that Caenorhabditis elegans lacking the homolog metl-5 develop phenotypes known to be associated with impaired translation. Altogether, our findings uncover critical and conserved roles of METTL5 in the regulation of translation.

N

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in mammalian mRNA. This modification is reversible and non-stoichiometric and adds another layer to the dynamic control of mRNA metabolism. The stability of m(6)A-modified mRNA is regulated by an m(6)A reader protein, human YTHDF2, which recognizes m(6)A and reduces the stability of target transcripts. Looking at additional functional roles for the modification, we find that another m(6)A reader protein, human YTHDF1, actively promotes protein synthesis by interacting with translation machinery. In a unified mechanism of m(6)A-based regulation in the cytoplasm, YTHDF2-mediated degradation controls the lifetime of target transcripts, whereas YTHDF1-mediated translation promotion increases translation efficiency, ensuring effective protein production from dynamic transcripts that are marked by m(6)A. Therefore, the m(6)A modification in mRNA endows gene expression with fast responses and controllable protein production through these mechanisms.

The prognosis for hepatocellular carcinoma (HCC) remains dismal in terms of overall survival (OS), and its molecular pathogenesis has not been completely defined. Here, we report that expression of deubiquitylase ubiquitin-specific protease 7 (USP7) is higher in human HCC tissues than in matched peritumoral tissues. Ectopic USP7 expression promotes growth of HCC cells in vivo and in vitro. Mechanistically, USP7 overexpression fosters HCC cell growth by forming a complex with and stabilizing thyroid hormone receptor-interacting protein 12 (TRIP12), which induces constitutive p14ARF ubiquitination. Clinically, USP7 overexpression is significantly correlated with a malignant phenotype, including larger tumor size, multiple tumor, poor differentiation, elevated alpha-fetoprotein, and microvascular invasion. Moreover, overexpression of USP7 and/or TRIP12 correlates with shorter OS and higher cumulative recurrence rates of HCC. Conclusion: USP7 stabilizes TRIP12 by deubiquitination, thus constitutively inactivating p14ARF and promoting HCC progression. This represents a novel marker for predicting prognosis and a potential therapeutic target for HCC. (Hepatology 2015;61:1603-1614)

N6-methyladenosine (m6A) is enriched in 3′untranslated region (3′UTR) and near stop codon of mature polyadenylated mRNAs in mammalian systems and has regulatory roles in eukaryotic mRNA transcriptome switch. Significantly, the mechanism for this modification preference remains unknown, however. Herein we report a characterization of the full m6A methyltransferase complex in HeLa cells identifying METTL3/METTL14/WTAP/VIRMA/HAKAI/ZC3H13 as the key components, and we show that VIRMA mediates preferential mRNA methylation in 3′UTR and near stop codon. Biochemical studies reveal that VIRMA recruits the catalytic core components METTL3/METTL14/WTAP to guide region-selective methylations. Around 60% of VIRMA mRNA immunoprecipitation targets manifest strong m6A enrichment in 3′UTR. Depletions of VIRMA and METTL3 induce 3′UTR lengthening of several hundred mRNAs with over 50% targets in common. VIRMA associates with polyadenylation cleavage factors CPSF5 and CPSF6 in an RNA-dependent manner. Depletion of CPSF5 leads to significant shortening of 3′UTR of over 2800 mRNAs, 84% of which are modified with m6A and have increased m6A peak density in 3′UTR and near stop codon after CPSF5 knockdown. Together, our studies provide insights into m6A deposition specificity in 3′UTR and its correlation with alternative polyadenylation.

N

N(6)-methyladenosine (m(6)A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export and translation. However, the precise m(6)A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc finger protein, plays an important role in modulating RNA m(6)A methylation in the nucleus. We show that knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m(6)A level on mRNA. Upon Zc3h13 knockdown a great majority of WTAP, Virilizer and Hakai translocate to the cytoplasm, suggesting that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m(6)A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer and Hakai in the nucleus to facilitate m(6)A methylation and to regulate mESC self-renewal.

N6-methyladenosine (m6A) is the most common internal modification in eukaryotic mRNA. It is dynamically installed and removed, and acts as a new layer of mRNA metabolism, regulating biological processes including stem cell pluripotency, cell differentiation, and energy homeostasis. m6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m6A-containing mRNAs, YTHDF2 expedites mRNA decay, and YTHDC1 affects the nuclear processing of its targets. The biological function of YTHDC2, the final member of the YTH protein family, remains unknown. We report that YTHDC2 selectively binds m6A at its consensus motif. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance. Ythdc2 knockout mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those of littermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and accordingly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m6A-binding protein that plays critical roles during spermatogenesis.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in cell differentiation and tissue development. It regulates multiple steps throughout the RNA life cycle including RNA processing, translation, and decay, via the recognition by selective binding proteins. In the cytoplasm, m6A binding protein YTHDF1 facilitates translation of m6A-modified mRNAs, and YTHDF2 accelerates the decay of m6A-modified transcripts. The biological function of YTHDF3, another cytoplasmic m6A binder of the YTH (YT521-B homology) domain family, remains unknown. Here, we report that YTHDF3 promotes protein synthesis in synergy with YTHDF1, and affects methylated mRNA decay mediated through YTHDF2. Cells deficient in all three YTHDF proteins experience the most dramatic accumulation of m6A-modified transcripts. These results indicate that together with YTHDF1 and YTHDF2, YTHDF3 plays critical roles to accelerate metabolism of m6A-modified mRNAs in the cytoplasm. All three YTHDF proteins may act in an integrated and cooperative manner to impact fundamental biological processes related to m6A RNA methylation.

N7-methylguanosine (m7G) is a positively charged, essential modification at the 5' cap of eukaryotic mRNA, regulating mRNA export, translation, and splicing. m7G also occurs internally within tRNA and rRNA, but its existence and distribution within eukaryotic mRNA remain to be investigated. Here, we show the presence of internal m7G sites within mammalian mRNA. We then performed transcriptome-wide profiling of internal m7G methylome using m7G-MeRIP sequencing (MeRIP-seq). To map this modification at base resolution, we developed a chemical-assisted sequencing approach that selectively converts internal m7G sites into abasic sites, inducing misincorporation at these sites during reverse transcription. This base-resolution m7G-seq enabled transcriptome-wide mapping of m7G in human tRNA and mRNA, revealing distribution features of the internal m7G methylome in human cells. We also identified METTL1 as a methyltransferase that installs a subset of m7G within mRNA and showed that internal m7G methylation could affect mRNA translation.

In the version of this article originally published, the references were incorrectly re-ordered during production. The hyphen in "N(6)-methyladenosine" in the title was also superscript. The errors have been corrected in the HTML and PDF versions of the paper.

UHRF1 (ubiquitin-like, with PHD and RING finger domains 1) is a critical epigenetic player involved in the maintenance of DNA methylation patterns during DNA replication. Dysregulation of the UHRF1 level is implicated in cancer onset, metastasis, and tumor recurrence. Previous studies demonstrated that UHRF1 can be stabilized through USP7-mediated deubiquitylation, but the mechanism through which UHRF1 is ubiquitylated is still unknown. Here we show that proteasomal degradation of UHRF1 is mediated by the SCF(β-TrCP) E3 ligase. Through bioinformatic and mutagenesis studies, we identified a functional DSG degron in the UHRF1 N terminus that is necessary for UHRF1 stability regulation. We further show that UHRF1 physically interacts with β-TrCP1 in a manner dependent on phosphorylation of serine 108 (S108(UHRF1)) within the DSG degron. Furthermore, we demonstrate that S108(UHRF1) phosphorylation is catalyzed by casein kinase 1 delta (CK1δ) and is important for the recognition of UHRF1 by SCF(β-TrCP). Importantly, we demonstrate that UHRF1 degradation is accelerated in response to DNA damage, coincident with enhanced S108(UHRF1) phosphorylation. Taken together, our data identify SCF(β-TrCP) as a bona fide UHRF1 E3 ligase important for regulating UHRF1 steady-state levels both under normal conditions and in response to DNA damage.

Transfer RNA (tRNA) is a central component of protein synthesis and cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and their decreased usage in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new regulatory mechanism of post-transcriptional gene expression.

TNF α plays a central role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and murine acute liver injury induced by injection of D-galactosamine and subsequent LPS. Recombinant Fc-fused soluble TNF receptor II (sTNFRII-Fc) has been used in the treatment of rheumatoid arthritis for a decade. We have recently constructed a novel fusion protein sTNFRII-gAD, which is composed of a soluble TNF receptor II and a globular domain of adiponectin. Utilizing the inclination of gAD to form homologous trimer naturally, we sought to explore TNFα antagonism of the novel trimerized sTNFRII-gAD and meantime compare TNFα-neutralizing effects in vitro and in vivo between sTNFRII-Fc and sTNFRII-gAD. Here, we evaluated the TNFα-antagonizing activity of sTNFRII-gAD with TNFα-induced L929 cytotoxicity assay. Furthermore, sTNFRII-Fc or sTNFRII-gAD was administered simultaneously with d-galactosamine 1h prior to LPS injection in the murine model of acute liver injury. Serum TNFα and TNFα-sTNFRII-gAD complex were measured by ELISA and the liver injury was assessed through alanine transaminase measurement and liver histological analysis. sTNFRII-gAD was shown to have higher TNFα-neutralizing activity than sTNFRII-Fc (p0.05) in the L929 cytotoxicity assay. With a significant attenuation of murine lethality (p0.05), sTNFRII-gAD showed more protective effects than sTNFRII-Fc in the murine model of acute liver injury. These results demonstrated that sTNFRII-gAD was more efficacious than sTNFRII-Fc as a TNFα antagonist, highlighting the potential of sTNFRII-gAD for the treatment of diseases associated with excessive TNFα.

Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHD(UHRF1)), an important regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHD(UHRF1) bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHD(UHRF1) binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.

Summary N 6 -methyladenosine (m 6 A) is an abundant modification in eukaryotic mRNA, regulating mRNA dynamics by influencing mRNA stability, splicing, export, and translation. However, the precise m 6 A regulating machinery still remains incompletely understood. Here we demonstrate that ZC3H13, a zinc-finger protein, plays an important role in modulating RNA m 6 A methylation in the nucleus. We show that knockdown of Zc3h13 in mouse embryonic stem cell significantly decreases global m 6 A level on mRNA. Upon Zc3h13 knockdown, a great majority of WTAP, Virilizer, and Hakai translocate to the cytoplasm, suggesting that Zc3h13 is required for nuclear localization of the Zc3h13-WTAP-Virilizer-Hakai complex, which is important for RNA m 6 A methylation. Finally, Zc3h13 depletion, as does WTAP, Virilizer, or Hakai, impairs self-renewal and triggers mESC differentiation. Taken together, our findings demonstrate that Zc3h13 plays a critical role in anchoring WTAP, Virilizer, and Hakai in the nucleus to facilitate m 6 A methylation and to regulate mESC self-renewal.

The histone H3 Lys 27 (H3K27) demethylase JMJD3 has been shown to play important roles in transcriptional regulation and cell differentiation. However, the mechanism underlying JMJD3-mediated transcriptional regulation remains incompletely understood. Here we show that JMJD3 is associated with KIAA1718, whose substrates include dimethylated H3K27 (H3K27me2), and proteins involved in transcriptional elongation. JMJD3 and KIAA1718 directly bind to and regulate the expression of a plethora of common target genes in both a demethylase activity-dependent and -independent manner in the human promyelocytic leukemia cell line HL-60. We found that JMJD3 and KIAA1718 collaborate to demethylate trimethylated H3K27 (H3K27me3) on a subset of their target genes, some of which are bivalently marked by H3K4me3 and H3K27me3 and associated with promoter-proximal, paused RNA polymerase II (Pol II) before activation. Reduction of either JMJD3 or KIAA1718 diminishes Pol II traveling along the gene bodies of the affected genes while having no effect on the promoter-proximal Pol II. Furthermore, JMJD3 and KIAA1718 also play a role in localizing elongation factors SPT6 and SPT16 to the target genes. Our results support the model whereby JMJD3 activates bivalent gene transcription by demethylating H3K27me3 and promoting transcriptional elongation. Taken together, these findings provide new insight into the mechanisms by which JMJD3 regulates gene expression.