Microbial Metabolite; Endogenous Metabolite

Human YTH domain family 2 (YTHDF2) proteins specifically identify N6-methyladenosine (m6A) and use it to control the degradation of mRNA. All eukaryotic messenger RNAs have N6-methyladenosine (m6A), an internal alteration that is widely present. M6A methyltransferases, such as MT-A70, install m6A post-transcriptionally on G(m6A)C(70%) or A within the consensus sequence (m6A)C (30%). N6-methyladenosine (m6A)-containing RNA is markedly reduced in the flow-through fraction and greatly enriched in the YTHDF-bound fraction [1]. The most prevalent internal RNA modification, N6-methyladenosine (m6A), is involved in many biological functions, including controlling the self-renewal and differentiation of embryonic stem cells. The catalytic subunits of methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) make up a portion of the large protein complex known as N6-methyladenosine (m6A) [2].

Our study demonstrated that cytokine CCL3, which is secreted by hepatocytes, promotes tumor metastasis by regulating m6A modification via vir-like m6A methyltransferase associated (VIRMA) in ICC cells. Moreover, immunohistochemical analyses showed that VIRMA correlated with poor outcomes in ICC patients. Finally, we confirmed both in vitro and in vivo that CCL3 could activate VIRMA and its critical downstream target SIRT1, which fuels tumor metastasis in ICC. Conclusions: In conclusion, our results enhanced our understanding of the interaction between hepatocytes and ICC cells, and revealed the molecular mechanism of the CCL3/VIRMA/SIRT1 pathway via m6A-mediated regulation in ICC metastasis. These studies highlight potential targets for the diagnosis, treatment, and prognosis of ICC. [4]

EMSA (Electrophoretic Mobility Shift Assay / Gel shift assay) [1]
The RNA probe was synthesized by a previously reported method with the sequence of 5’-AUGGGCCGUUCAUCUGCUAAAAGGXCUGCUUUUGGGGCUUGU-3’(X = A or m6A). After the synthesis, the RNA probe was labeled in a reaction mixture of 2 µL RNA probe (1 µM), 5 µL 5×T4 PNK buffer A (Fermentas), 1 µL T4 PNK (Fermentas), 1 µL32P-ATP and 41 µL RNase-free water (final RNA concentration 40 nM) at 37°C for 1 hour. The mixture was then purified by RNase-free micro bio-spin columns with bio-gel P30 in Tris buffer (BioRad 732–6250) to remove hot ATP and other small molecules. To the elute, 2.5 µL 20 × SSC buffer was added. The mixture was heated to 65°C for 10 min to denature the RNA probe, and then slowly cooled down to room temperature. GST-YTHDF1–3 were diluted to concentration series of 200 nmol, 1 µM, 5 µM, 20 µM and 100 µM (or other indicated concentrations) in binding buffer (10 mM HEPES, pH 8.0, 50 mM KCl, 1mM EDTA, 0.05% Triton-X-100, 5% glycerol, 10 µg/mL Salmon DNA, 1 mM DTT and 40 U/mL RNasin). Before loading to each well, 1 µL RNA probe (4 nM final concentration) and 1 µL protein (20 nM, 100 nM, 500 nM, 2 µM, or 10 µM final concentration) were added and the solution was incubated on ice for 30 min. The entire 10 µL RNA-protein mixture was loaded to the gel (Novex 4~20% TBE gel) and run at 4 °C for 90 min at 90 V. Quantification of each band was carried out by using a storage phosphor screen (K-Screen; Fuji film) and Bio-Rad Molecular Imager FX in combination with Quantity One software (Bio-Rad). The Kd (dissociation constant) was calculated with nonlinear curve fitting (Function Hyperbl) of Origin 8 software with y = P1×x/(P2+x), where y is the ratio of [RNA-protein]/[free RNA]+[RNA-protein], x is the concentration of the protein, and P2 is Kd.

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In vitro pull down [1]
0.8 µg mRNA (save 0.2 µg from the same sample as input) and YTHDF1–3 or C-YTHDF2 (final concentration 500 nM) were diluted into 200 µL IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris, pH 7.4, 40 U/mL RNase inhibitor, 0.5 mM DTT), and the solution was mixed with rotation at 4 °C for 2 h. For YTHDF1–3, 10 µL GST-affinity magnetic beads (Pierce) were used for each sample after being washed four times with 200 µL IPP buffer for each wash. For C-YTHDF2, 20 µL Dynabeads® His-Tag Isolation & Pulldown beads (Invitrogen) were used after being washed four times with 200 µL IPP buffer for each wash. The beads were then re-suspended in 50 µL IPP buffer. The protein-RNA mixture was combined with GST or His6 beads and kept rotating for another 2 h at 4 °C. The aqueous phase was collected, recovered by ethanol precipitation, dissolved in 15 µL water, and saved as the flowthrough. The beads were washed four times with 300 µL IPP buffer each time. 0.4 mL trizol reagent was added to the beads and further purified according to manufacturer’s instruction. The purified fraction was dissolved in 15 µL water, and saved as YTHDF-bound. LC-MS/MS was used to measure the level of m6A in each sample of input, flowthrough, and YTHDF-bound.

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LC-MS/MS [1]
200–300 ng of mRNA was digested by nuclease P1 (2 U) in 25 µl of buffer containing 25 mM of NaCl, and 2.5 mM of ZnCl2 at 37 °C for 2 h, followed by the addition of NH4HCO3 (1 M, 3 µl) and alkaline phosphatase (0.5 U). After an additional incubation at 37 °C for 2 h, the sample was diluted to 50 µL and filtered (0.22 µm pore size, 4 mm diameter, Millipore), and 5 µl of the solution was injected into LC-MS/MS. Nucleosides were separated by reverse phase ultra-performance liquid chromatography on a C18 column with on-line mass spectrometry detection using Agilent 6410 QQQ triple-quadrupole LC mass spectrometer in positive electrospray ionization mode. The nucleosides were quantified by using the nucleoside to base ion mass transitions of 282 to 150 (m6A), and 268 to 136 (A). Quantification was performed in comparison with the standard curve obtained from pure nucleoside standards running on the same batch of samples. The ratio of m6A to A was calculated based on the calibrated concentrations.

m6A profiling [1]
Total RNA was isolated from HeLa cells with TRIZOL reagent. Poly(A)+ RNA was further enriched from total RNA by using FastTrack MAG Maxi mRNA isolation kit. In particularly, an additional DNase I digestion step was applied to all the samples to avoid DNA contamination. RNA fragmentation, m6A-seq, and library preparation were performed according to the previous protocol developed by Dominissini et al14. The experiment was conducted in two biological replicates (Extended Data Table 1).

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N(6)-methyladenosine (m(6)A), the most abundant internal RNA modification, functions in diverse biological processes, including regulation of embryonic stem cell self-renewal and differentiation. As yet, methods to detect m(6)A in the transcriptome rely on the availability and quality of an m(6)A antibody and are often associated with a high rate of false positives. Here, based on our observation that m(6)A interferes with A-T/U pairing, we report a microarray-based technology to map m(6)A sites in mouse embryonic stem cells. We identified 72 unbiased sites exhibiting high m(6)A levels from 66 PolyA RNAs. Bioinformatics analyses suggest identified sites are enriched on developmental regulators and may in some contexts modulate microRNA/mRNA interactions. Overall, we have developed microarray-based technology to capture highly enriched m(6)A sites in the mammalian transcriptome. This method provides an alternative means to identify m(6)A sites for certain applications. [2]

Female Balb/c nude mice (Balb/c-nu, weighing ~ 15–20 g), were used. To generate a subcutaneous xenograft tumor model, we injected 5 × 106 transfected HuCCT1 cells, which were suspended in 0.2 ml of PBS, into the flank of nude mice (five mice per group). On the other hand, an orthotopic xenograft tumor model was established by inoculating 3 × 106 transfected HuCCT1 cells in 50 μl of PBS into the liver of nude mice (five mice per group). For the treated groups, HuCCT1 cells were treated with CCL3 (100 ng/ml) for 6 h before inoculation. For subcutaneous injection, the intratumoral multiple-point injection of CCL3 (20 mg/kg) diluted in 50 μl PBS was performed every 5 days. The control groups were treated with PBS. Subcutaneous tumor size was measured twice a week. After in vivo fluorescence imaging, all the study mice were sacrificed with the in vivo imaging system (IVIS) spectrum after four weeks. The mice tumors and organs were dissected, photographed, weighed, and stained. [4]

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Background: Intrahepatic cholangiocarcinoma (ICC) is a malignant disease characterized by onset occult, rapid progression, high relapse rate, and high mortality. However, data on how the tumor microenvironment (TME) regulates ICC metastasis at the transcriptomic level remains unclear. This study aimed to explore the mechanisms and interactions between hepatocytes and ICC cells. Methods: We analyzed the interplay between ICC and liver microenvironment through cytokine antibody array analysis. Then we investigated the role of N6-methyladenosine (m6A) modification and the downstream target in vitro, in vivo experiments, and in clinical specimens. [4]

[1]. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014 Jan 2;505(7481):117-20.

[2]. Genome-wide detection of high abundance N6-methyladenosine sites by microarray. RNA. 2015 Aug;21(8):1511-8.

[3]. N6-Methyladenosine and Viral Infection. Front Microbiol. 2019 Mar 5;10:417.

[4]. CCL3 secreted by hepatocytes promotes the metastasis of intrahepatic cholangiocarcinoma by VIRMA-mediated N6-methyladenosine (m6A) modification. J Transl Med. 2023 Jan 23;21(1):43.

N(6)-methyladenosine is a methyladenosine compound with one methyl group attached to N(6) of the adenine nucleobase.

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N6-methyladenosine (m6A), as a dynamic posttranscriptional RNA modification, recently gave rise to the field of viral epitranscriptomics. The interaction between virus and host is affected by m6A. Multiple m6A-modified viral RNAs have been observed. The epitranscriptome of m6A in host cells are altered after viral infection. The expression of viral genes, the replication of virus and the generation of progeny virions are influenced by m6A modifications in viral RNAs during virus infection. Meanwhile, the decorations of m6A in host mRNAs can make viral infections more likely to happen or can enhance the resistance of host to virus infection. However, the mechanism of m6A regulation in viral infection and host immune response has not been thoroughly elucidated to date. With the development of sequencing-based biotechnologies, transcriptome-wide mapping of m6A in viruses has been achieved, laying the foundation for expanding its functions and corresponding mechanisms. In this report, we summarize the positive and negative effects of m6A in distinct viral infection. Given the increasingly important roles of m6A in diverse viruses, m6A represents a novel potential target for antiviral therapy. [3]

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In our study, we only employed the m6A dot blot assay to quantify the m6A levels of ICC, and showed that SIRT1 is a downstream target of VIRMA. Thus, there is a need for further studies which would take the following variables into account: (1) examine the m6A levels of ICC using a colorimetric strategy or liquid chromatography-mass spectrometry (LC–MS), (2) detect whether VIRMA functions independently of its m6A catalytic activity in cancer progression, and (3) develop a peptide inhibitor to target VIRMA domain and explore whether it may be beneficial in the treatment of ICC.
In conclusion, our study highlights the critical role of VIRMA-mediated m6A modification in ICC progression and metastasis. Mechanistically, we demonstrated that CCL3 is secreted by hepatocytes and may promote metastasis of ICC cells by regulating m6A methylation. The regulation of m6A methylation is mediated by VIRMA, which epigenetically promotes SIRT1 expression through an m6A methylation-dependent mechanism. Our results suggested that the interaction between hepatocytes and ICC cells might offer a possible interventional target for ICC. Besides, the m6A modification on tumor metastasis will contribute to further studies that would explore molecular mechanisms and identify efficient treatment strategies against ICC. [4]

C11H15N5O4

281.27

281.112

C, 46.97; H, 5.38; N, 24.90; O, 22.75

1867-73-8

N6-Methyladenosine-d3;139896-43-8

102175

White to off-white solid powder

1.85g/cm3

649.1ºC at 760mmHg

346.3ºC

9.86E-18mmHg at 25°C

1.814

-0.4

4

8

3

20

349

4

CNC1=C2C(=NC=N1)N(C=N2)[C@H]3[C@@H]([C@@H]([C@H](O3)CO)O)O

VQAYFKKCNSOZKM-IOSLPCCCSA-N

InChI=1S/C11H15N5O4/c1-12-9-6-10(14-3-13-9)16(4-15-6)11-8(19)7(18)5(2-17)20-11/h3-5,7-8,11,17-19H,2H2,1H3,(H,12,13,14)/t5-,7-,8-,11-/m1/s1

(2R,3S,4R,5R)-2-(hydroxymethyl)-5-[6-(methylamino)purin-9-yl]oxolane-3,4-diol

6-Methylaminopurinosine; N6-Methyladenosine; m6A; 1867-73-8; N-Methyladenosine; 6-Methyladenosine; Adenosine, N-methyl-; N(6)-Methyladenosine; 6-Methylaminopurinosine; 6-Methylaminopurine riboside;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 31 mg/mL (~110.21 mM)
H2O : ~5.56 mg/mL (~19.77 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.40 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.40 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)