METTL3/N6-methyladenosine/ miR-21-5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF-κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR-21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF-kB signalling pathway. However, whether miR-21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6-methyladenosine (m⁶A) modification-dependent primary microRNA (pri-microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR-21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK-2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR-21-5p mimic or miR-21-5p inhibitor. We found that the levels of miR-21-5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF-kB pathway was activated by miR-21-5p, confirming that miR-21-5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR-21-5p maturation. Our research is the first to demonstrate the role of the METTL3-m⁶A-miR-21-5p-SPRY1/ERK/NF-kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

METTL3 promotes prostatic hyperplasia by regulating PTEN expression in an m6A-YTHDF2-dependent manner

Uncontrolled epithelial cell proliferation in the prostate transition zone and the hyper-accumulation of mesenchymal-like cells derived from the epithelial-mesenchymal transition (EMT) of prostatic epithelium are two key processes in benign prostatic hyperplasia (BPH). m⁶A RNA modification affects multiple cellular processes, including cell proliferation, apoptosis, and differentiation. In this study, the aberrant up-regulation of methylase METTL3 in BPH samples suggests its potential role in BPH development. Elevated m⁶A modification in the prostate of the BPH rat was partially reduced by METTL3 knockdown. METTL3 knockdown also partially reduced the prostatic epithelial thickness and prostate weight, significantly improved the histological features of the prostate, inhibited epithelial proliferation and EMT, and promoted apoptosis. In vitro, METTL3 knockdown decreased TGF-β-stimulated BPH-1 cell proliferation, m⁶A modification, and EMT, whereas promoted cell apoptosis. METTL3 increased the m⁶A modification of PTEN and inhibited its expression through the reading protein YTHDF2. PTEN knockdown aggravated the molecular, cellular, and pathological alterations in the prostate of BPH rats and amplified TGF-β-induced changes in BPH-1 cells. More importantly, PTEN knockdown partially abolished the improving effects of METTL3 knockdown both in vivo and in vitro. In conclusion, the level of m⁶A modification is elevated in BPH; the METTL3/YTHDF2/PTEN axis disturbs the balance between epithelial proliferation and apoptosis, promotes EMT, and accelerates BPH development in an m⁶A modification-related manner.Subject terms: Cell biology, Molecular biology     

p53 m6A modulation sensitizes hepatocellular carcinoma to apatinib through apoptosis

Hepatocellular carcinoma (HCC) is insidious and prone to metastasis and recurrence. Currently, no effective treatment is available for HCC. Furthermore, HCC does not respond to various radio- and chemotherapies, and the molecular mechanism of treatment resistance is unclear. Here, we found that p53 n6-methyladenosine (m⁶A) played a decisive role in regulating HCC sensitivity to chemotherapy via the p53 activator RG7112 and the vascular endothelial growth factor receptor inhibitor apatinib. Our results reveal that p53 activation plays a crucial role in chemotherapy-induced apoptosis and reducing cell viability. Moreover, decreasing m⁶A methyltransferase (e.g., methyltransferase-like 3, METTL3) expression through chemotherapeutic drug combinations reduced p53 mRNA m⁶A modification. p53 mRNA m⁶A modification blockage induced by S-adenosyl homocysteine or siRNA-mediated METTL3 inhibition enhanced HCC sensitivity to chemotherapy. Importantly, we observed that downregulation of METTL3 and upregulation of p53 expression by oral administration of chemotherapy drugs triggered apoptosis and xenograft tumor growth inhibition in nude mice. Based on these findings, we hypothesize that a METTL3–m⁶A–p53 axis could be a potential target in HCC therapy.

     

Molecular basis for human mitochondrial tRNA m3C modification by alternatively spliced METTL8

METTL8 has recently been identified as the methyltransferase catalyzing 3-methylcytidine biogenesis at position 32 (m³C32) of mitochondrial tRNAs. METTL8 also potentially participates in mRNA methylation and R-loop biogenesis. How METTL8 plays multiple roles in distinct cell compartments and catalyzes mitochondrial tRNA m³C formation remain unclear. Here, we discovered that alternative mRNA splicing generated several isoforms of METTL8. One isoform (METTL8-Iso1) was targeted to mitochondria via an N-terminal pre-sequence, while another one (METTL8-Iso4) mainly localized to the nucleolus. METTL8-Iso1-mediated m³C32 modification of human mitochondrial tRNA^Thr (hmtRNA^Thr) was not reliant on t⁶A modification at A37 (t⁶A37), while that of hmtRNA^Ser(UCN) critically depended on i⁶A modification at A37 (i⁶A37). We clarified the hmtRNA^Thr substrate recognition mechanism, which was obviously different from that of hmtRNA^Ser(UCN), in terms of requiring a G35 determinant. Moreover, SARS2 (mitochondrial seryl-tRNA synthetase) interacted with METTL8-Iso1 in an RNA-independent manner and modestly accelerated m³C modification activity. We further elucidated how nonsubstrate tRNAs in human mitochondria were efficiently discriminated by METTL8-Iso1. In summary, our results established the expression pattern of METTL8, clarified the molecular basis for m³C32 modification by METTL8-Iso1 and provided the rationale for the involvement of METTL8 in tRNA modification, mRNA methylation or R-loop biogenesis.     

METTL3/N6‐methyladenosine/ miR‐21‐5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF‐κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR‐21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF‐kB signalling pathway. However, whether miR‐21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6‐methyladenosine (m⁶A) modification‐dependent primary microRNA (pri‐microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR‐21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK‐2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR‐21‐5p mimic or miR‐21‐5p inhibitor. We found that the levels of miR‐21‐5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF‐kB pathway was activated by miR‐21‐5p, confirming that miR‐21‐5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR‐21‐5p maturation. Our research is the first to demonstrate the role of the METTL3‐m⁶A‐miR‐21‐5p‐SPRY1/ERK/NF‐kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

Methyltransferase-like 3-mediated N6-methyladenosine modification of miR-7212-5p drives osteoblast differentiation and fracture healing

N6-methyladenosine (m6A) modification has been reported in various diseases and implicated in increasing numbers of biological processes. However, previous studies have not focused on the role of m6A modification in fracture healing. Here, we demonstrated that m6A modifications are decreased during fracture healing and that methyltransferase-like 3 (METTL3) is the main factor involved in the abnormal changes in m6A modifications. Down-regulation of METTL3 promotes osteogenic processes both in vitro and in vivo, and this effect is recapitulated by the suppression of miR-7212-5p maturation. Further studies have shown that miR-7212-5p inhibits osteoblast differentiation in MC3T3-E1 cells by targeting FGFR3. The present study demonstrated an important role of the METTL3/miR-7212-5p/FGFR3 axis and provided new insights on m6A modification in fracture healing.     

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.     

Low METTL3 level in midgut of the Bombyx mori inhibit the proliferation of nucleopolyhedrovirus

N6-methyladeosine (m⁶A) plays an important role in virus infection and replication. Bombyx mori nuclear polyhedrosis is caused by Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Expression levels of m⁶A-modification-related genes after the infection of BmNPV were detected at first. Then, expression levels of BmNPV nucleocapsid protein gene VP39 and envelope fusion protein gene GP64 after knockdown of METTL3in vitro were quantified to identify the effect of m⁶A modification on BmNPV. BmNPV firstly infects the larval midgut in case of oral infection. Subsequently, to clarify the relationship between m⁶A modification and resistance of the silkworm to BmNPV, we detected the expression levels of m⁶A-modification-related genes invivo before and after infection of BmNPV. The results indicated that low METTL3 level hindered the proliferation of BmNPV to some extent, and silkworm strain with low METTL3 level showed stronger resistance against BmNPV. This study will accumulate new experimental data for elucidating the resistance mechanism of silkworm against BmNPV.     

Loss of Wtap results in cerebellar ataxia and degeneration of Purkinje cells

N⁶-methyladenosine (m⁶A) modification, which is achieved by the METTL3/METTL14/WTAP methyltransferase complex, is the most abundant internal mRNA modification. Although recent evidence indicates that m⁶A can regulate neurodevelopment as well as synaptic function, the roles of m⁶A modification in the cerebellum and related synaptic connections are not well established. Here, we report that Purkinje cell (PC)-specific WTAP knockout mice display early-onset ataxia concomitant with cerebellar atrophy due to extensive PC degeneration and apoptotic cell death. Loss of Wtap also causes the aberrant degradation of multiple PC synapses. WTAP depletion leads to decreased expression levels of METTL3/14 and reduced m⁶A methylation in PCs. Moreover, the expression of GFAP and NF-L in the degenerating cerebellum is increased, suggesting severe neuronal injuries. In conclusion, this study demonstrates the critical role of WTAP-mediated m⁶A modification in cerebellar PCs, thus providing unique insights related to neurodegenerative disorders.     

Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

A subset of eukaryotic tRNAs is methylated in the anticodon loop, forming 3-methylcytosine (m³C) modifications. In mammals, the number of tRNAs containing m³C modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing m³C formation in nuclear-encoded tRNAs have been identified, the proteins responsible for m³C modification in mt-tRNAs are unknown. Here, we show that m³C formation in human mt-tRNAs is dependent upon the methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase, as well as with mt-tRNAs containing m³C. We demonstrate that human cells deficient in METTL8 exhibit loss of m³C modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m³C in METTL8-deficient cells could be rescued by re-expression of WT METTL8, but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, we found METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA, suggesting a role for m³C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m³C formation in mt-tRNAs and uncover a potential function for m³C modification in mitochondrial tRNA structure.     

m6A甲基转移酶家族Fiona/METT-10/METTL16的功能

RNA碱基上的化学修饰在其功能的精准调节中发挥关键作用,其中m⁶A是自然界中最普遍的RNA修饰之一,且该修饰在调控RNA稳定性、pre-mRNA剪接、翻译等方面具有重要功能。在真核生物中,m⁶A修饰主要由两种甲基转移酶完成,其在哺乳动物中分别命名为METTL3和METTL16。与METTL3相似,METTL16的底物多种多样,包括pre-mRNA、rRNA、snRNA和lncRNA等,因此似乎难以用一种分子机理解释METTL16对不同RNA底物进行m⁶A修饰的功能。此外,METTL16还在翻译调控中发挥重要作用,但此过程不依赖其甲基转移酶活性,这进一步增加了高度保守的METTL16的功能复杂性。本综述总结了METTL16及其同源蛋白质的结构域、甲基化底物以及它们的潜在功能,着重阐述了在不同物种中关于METTL16研究结果的矛盾之处,并推测METTL16调控S-腺苷基甲硫氨酸(SAM)代谢的功能是趋同进化的一个潜在案例。     

Deregulation of neuronal miRNAs induced by amyloid-β or TAU pathology

Background

Despite diverging levels of amyloid-β (Aβ) and TAU pathology, different mouse models, as well as sporadic AD patients show predictable patterns of episodic memory loss. MicroRNA (miRNA) deregulation is well established in AD brain but it is unclear whether Aβ or TAU pathology drives those alterations and whether miRNA changes contribute to cognitive decline.

Methods

miRNAseq was performed on cognitively intact (4 months) and impaired (10 months) male APPtg (APP^swe/PS1^L166P) and TAUtg (THY-Tau22) mice and their wild-type littermates (APPwt and TAUwt). We analyzed the hippocampi of 12 mice per experimental group (n =?96 in total), and employed a 2-way linear model to extract differentially expressed miRNAs. Results were confirmed by qPCR in a separate cohort of 4 M and 10 M APPtg and APPwt mice (n =?7–9 per group) and in human sporadic AD and non-demented control brain. Fluorescent in situ hybridization identified their cellular expression. Functional annotation of predicted targets was performed using GO enrichment. Behavior of wild-type mice was assessed after intracerebroventricular infusion of miRNA mimics.

Results

Six miRNAs (miR-10a-5p, miR-142a-5p, miR-146a-5p, miR-155-5p, miR-211-5p, miR-455-5p) are commonly upregulated between APPtg and TAUtg mice, and four of these (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) are altered in AD patients. All 6 miRNAs are strongly enriched in neurons. Upregulating these miRNAs in wild-type mice is however not causing AD-related cognitive disturbances.

Conclusion

Diverging AD-related neuropathologies induce common disturbances in the expression of neuronal miRNAs. 4 of these miRNAs are also upregulated in AD patients. Therefore these 4 miRNAs (miR-142a-5p, miR-146a-5p, miR-155-5p and miR-455-5p) appear part of a core pathological process in AD patients and APPtg and TAUtg mice. They are however not causing cognitive disturbances in wild-type mice. As some of these miRNA target AD relevant proteins, they may be, in contrast, part of a protective response in AD.

     

METTL3‐mediated maturation of miR‐589‐5p promotes the malignant development of liver cancer

MiR‐589‐5p could promote liver cancer, but the specific mechanisms are largely unknown. This study examined the role and mechanisms of miR‐589‐5p in liver cancer. The expressions of miR‐589‐5p, METTL3 and m6A in liver cancers were determined by RT‐qPCR. The relationship between miR‐589‐5p and METTL3‐mediated m6A methylation was examined by m6A RNA immunoprecipitation. After transfection, the viability, migration, invasion and expressions of METTL3 and miR‐589‐5p in liver cancer cells were detected by CCK‐8, wound‐healing, transwell and RT‐qPCR. After the xenograft tumour was established in mice, the tumour volume was determined and the expressions of METTL3, miR‐589‐5p, MMP‐2, TIMP‐2, E‐cadherin, N‐cadherin and Vimentin in tumour tissue were detected by RT‐qPCR and Western blotting. In vitro study showed that miR‐589‐5p and METTL3 were highly expressed in liver cancer. METTL3 was positively correlated with miR‐589‐5p. METTL3 up‐regulated the expression of miR‐589‐5p and promoted the maturation of miR‐589‐5p. Overexpressed miR‐589‐5p and METTL3 promoted the viability, migration and invasion of liver cancer cells, while the effects of silencing miR‐589‐5p and METTL3 on the cells were the opposite. The effects of METTL3 overexpression and silencing were reversed by miR‐589‐5p inhibitor and mimic, respectively. In vivo study showed that METLL3 silencing inhibited the growth of xenograft tumour and the expressions of METTL3, MMP‐2, N‐cadherin and Vimentin, promoted the expressions of TIMP‐2 and E‐cadherin, while miR‐589‐5p mimic caused the opposite results and further reversed the effects of METLL3 silencing. In summary, this study found that METTL3‐mediated maturation of miR‐589‐5p promoted the malignant development of liver cancer.