N^6-methyladenosine （m^6A） is the most common internal modification in eukaryotic mRNA. It is dynamically in- stalled and removed, and acts as a new layer of mRNA metabolism, regulating biological ...processes including stem cell pluripotency, cell differentiation, and energy homeostasis, m^6A is recognized by selective binding proteins; YTHDF1 and YTHDF3 work in concert to affect the translation of m^6A-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 knock- out mice are infertile; males have significantly smaller testes and females have significantly smaller ovaries compared to those oflittermates. The germ cells of Ythdc2 knockout mice do not develop past the zygotene stage and according- ly, Ythdc2 is upregulated in the testes as meiosis begins. Thus, YTHDC2 is an m6A-binding protein that plays critical roles during svermatogenesis.
Abstract
Kinase-catalyzed phosphorylation plays a crucial role in pathological cardiac hypertrophy. Here, we show that CDC-like kinase 4 (CLK4) is a critical regulator of cardiomyocyte hypertrophy ...and heart failure. Knockdown of
Clk4
leads to pathological cardiomyocyte hypertrophy, while overexpression of
Clk4
confers resistance to phenylephrine-induced cardiomyocyte hypertrophy. Cardiac-specific
Clk4
-knockout mice manifest pathological myocardial hypertrophy with progressive left ventricular systolic dysfunction and heart dilation. Further investigation identifies nexilin (NEXN) as the direct substrate of CLK4, and overexpression of a phosphorylation-mimic mutant of NEXN is sufficient to reverse the hypertrophic growth of cardiomyocytes induced by
Clk4
knockdown. Importantly, restoring phosphorylation of NEXN ameliorates myocardial hypertrophy in mice with cardiac-specific
Clk4
deletion. We conclude that CLK4 regulates cardiac function through phosphorylation of NEXN, and its deficiency may lead to pathological cardiac hypertrophy. CLK4 is a potential intervention target for the prevention and treatment of heart failure.
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.
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 (PHDUHRF1), 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 PHDUHRF1 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 PHDUHRF1 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.
► In vitro binding and ITC identify PHDUHRF1 domain as an unmodified histone H3R2 reader ► Crystal structure shows hydrogen bonding, electrostatics, and hydrophobic interactions ► Genome-wide analyses demonstrate a general transcriptional role for UHRF1 ► Binding unmodified H3R2 is critical for UHRF1 to regulate gene expression
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.
Abstract
Dynamic mRNA modification in the form of
N
6
-methyladenosine (m
6
A) adds considerable richness and sophistication to gene regulation. The m
6
A mark is asymmetrically distributed along ...mature mRNAs, with approximately 35% of m
6
A residues located within the coding region (CDS). It has been suggested that methylation in CDS slows down translation elongation. However, neither the decoding feature of endogenous mRNAs nor the physiological significance of CDS m
6
A has been clearly defined. Here, we found that CDS m
6
A leads to ribosome pausing in a codon-specific manner. Unexpectedly, removing CDS m
6
A from these transcripts results in a further decrease of translation. A systemic analysis of RNA structural datasets revealed that CDS m
6
A positively regulates translation by resolving mRNA secondary structures. We further demonstrate that the elongation-promoting effect of CDS methylation requires the RNA helicase-containing m
6
A reader YTHDC2. Our findings established the physiological significance of CDS methylation and uncovered non-overlapping function of m
6
A reader proteins.
UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to ...contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.
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, m~A binding protein YTHDF1 facilitates translation of m6A-modified mRNAs, and YTHDF2 acceler- ates 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 YTHDFI, and affects methylated mRNA decay mediated through YTHDF2. Cells defcient 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.
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•Presence of internal m7G in mammalian mRNA•Chemical-assisted m7G-seq maps m7G methylome at single-base resolution•Highly modified internal m7G sites were identified in human mRNA•METTL1 acts as a methyltransferase for a subset of internal m7G sites in mRNA
Zhang et al. discovered the presence of internal N7-methylguanosine (m7G) within mammalian mRNA. Both antibody-based and chemical-assisted methods were developed for transcriptome-wide mapping of internal m7G, with the latter reaching single-base resolution. METTL1/WDR4 was identified as a writer complex that installs a subset of m7G on mRNA, which affects translation.
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