N6-Methyladenosine is an abundant nucleoside in cellular mRNA that undergoes demethylation under physiological conditions by fat mass.
Mammalian cell culture, siRNAknockdown and cell fractionation
Human cervical adenocarcinoma cell line HeLa (CCL-2) and human embryonic kidney (HEK) cell line HEK293T (CRL-11268) were cultured under standard conditions. Control siRNA, METTL3 siRNA, METTL14 siRNA or HNRNPG siRNA was transfected into HEK293T cells at a concentration of 20–50 nM, using Lipofectamine RNAiMAX. Cells were collected 48 h after transfection, shock-frozen in liquid nitrogen and stored at−80◦C for further studies. Nuclear and cytoplasmic extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction Reagents
10–30μg protein samples were separated on 4–12% polyacrylamide Bis-Tris gels and transferred to polyvinylidene fluoride membranes. The blots were probed with METTL3-, METTL14-, HNRNPG- or GAPDH- specific primary antibody, followed by rabbit antigoat IgG-HRP or goat anti-rabbit IgG-HRP secondary antibody, and then visualized by enhanced chemoluminescence.
For expression of the N-terminal RNA recognition motif (N-RRM, residues 1–83) and C-terminal RNA binding domain (C-RBD, residues 334–391) of human HNRNPG protein, sequences encoding these HNRNPG domains were amplified by polymerase chain reaction (PCR) from human HeLa cDNA libraries and then subcloned into pGEX-6p-1 expression vectors using BamHI andXhoI restriction sites. PlasmidDNAwas transformed into Escherichia coli BL21-CodonPlus(DE3)-RP or BL21-CodonPlus(DE3)-RIL cells. The transformed bacteria were grown to saturation at 37oC, 200 rpm in Luria–Bertani Lennox medium with 100μg/ml ampicillin, then diluted 1:100, grown in the same culturemedium to an absorbance of∼0.6 at 600 nm, and induced with 1 mM isopropylβ-D-1-thiogalactoside (IPTG). The bacteria were grown an additional 16–22 h at 18oC, 200 rpm, then harvested and sonicated at 4◦C. GST-fusion proteins were isolated fromthe soluble lysate using glutathione-Sepharose beads and stored in 10 mM Tris-Cl (pH 7.4), 100 mM KCl, 2.5 mM MgCl2, 30% glycerol at −80oC.
The following RNA oligos were synthesized2by Q.D. and purified by high-performance liquid chromatography (HPLC) and/or denaturing gel electrophoresis.
The RNA oligos used:
2,515-A: 5’- AAUGUGAAGGACUUUCGUAACGGAAGUAAUUCAA-Biotin;
2,515-m6A: 5’- AAUGUGAAGGm6ACUUUCGUAACGGAAGUAAUUCAA-Biotin.
RNA pull-down, gel shift and cross-linking
The eluted protein samples were separated on 4–12% polyacrylamide Bis-Tris gelsand stained with SYPRO Ruby Protein Gel Stain according to themanufacturer’s instructions. Proteins in gel slices or in the entire pulled-down protein sample were digested with trypsin and identified using LC–MS/MS by the Donald Danforth Plant Science Center.
The gel-purified 5’32Plabeled RNA oligos were refolded by heating at 90oC for 1 min, then at 30oC for 5 min. A total of 3μl nuclear extract and 6μl refolded RNA were incubated together at room temperature for 30 min, and then at 4oC for 2 h. Each sample was mixed with 1μl 50% glycerol, separated on an 8% polyacrylamide, 44.5 mMTris–borate (pH 8.3 at 25oC, pH 8.9 at 5oC), and 1 mM Na2EDTA (ethylenediaminetetraacetic acid) native gel at 5 ◦C, and visualized by phosphorimaging using the Personal Molecular Imager.
动物 A (mg/kg) = 动物 B (mg/kg)×动物 B的Km系数/动物 A的Km系数
例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。
 Liu N, Zhou KI, Parisien M, Dai Q, Diatchenko L, Pan T. N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein. Nucleic Acids Res. 2017.