细胞实验
Mammalian cell culture, siRNAknockdown and cell fractionation[1]
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
Western blotting
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.
Protein expression
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.
RNA oligos
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.
