Fig 1.
Immuno-northern blotting protocol.
In this method, RNAs were separated by denaturing acrylamide or agarose gel electrophoresis, transferred onto a positively charged nylon membrane followed by UV cross-linking, and then incubated with the primary antibodies against the specific modified nucleoside as well as the secondary antibody. The specific bands were visualized by chemiluminescence.
Fig 2.
Immuno-northern blotting using antibodies against modified nucleosides.
(A) Structural formulas of 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine (Ψ), and 5-methylcytidine (m5C). (B) Total RNA (0.6 μg) isolated from the indicated samples was separated in the 12% polyacrylamide-8 M urea denaturing gel and analyzed by SYBR staining and immuno-northern blotting (INB) using the anti-m1A, anti-m6A, anti-Ψ and anti-m5C antibodies. The total RNA samples (0.6 μg) were also analyzed by the dot blot assay using each antibody, as indicated in the bottom panels.
Fig 3.
Immuno-northern blot analysis using the anti-m5C antibody with acrylamide and agarose gel separation.
(A) Total RNA (0.6 μg) isolated from mouse liver was analyzed by SYBR staining and INB with anti-m5C antibody after electrophoresis in the 12% polyacrylamide or 1% agarose denaturing gel. The INB image using acrylamide gel is a long-exposure image. Arrowheads denote the positive signals, which appear to be integrated in the INB image using the acrylamide gel. (B) Total RNA isolated from mouse liver (1.2 μg) and E. coli HST04 (0.6 μg) was analyzed by SYBR staining and INB with anti-m5C antibody or isotype IgG. As a negative control for INB, isotype IgG was used instead of anti-m5C antibody at the same concentration. (C, D) The effect of DNase I treatment on the anti-m5C positive signal. Total RNAs isolated from mouse liver (1.2 μg, C), E. coli HST04 (0.6 μg, D), and yeast BY4742 (1.0 μg, D) were treated with or without DNase I, and then it was analyzed by SYBR staining and INB with anti-m5C antibody using the indicated gel. Arrowheads denote the positive signals.
Fig 4.
Immuno-northern blot analysis of mitochondrial and non-mitochondrial RNA.
RNAs were isolated from the total fraction (Total), mitochondrial fraction (Mito.), and non-mitochondrial fraction (Non-mito.) of mouse liver. (A) Each total RNA (0.6 μg) was separated in the 12% polyacrylamide gel and analyzed by SYBR staining and INB with anti-m1A antibody. The red arrowheads denote shorter tRNA bands, which were rich in the mitochondrial RNA. Arrows denote slight anti-m1A positive bands in the long exposure image. White arrowheads denote negative signals in 5.8S and 5S rRNA. (B) Each total RNA (1.2 μg) was separated on the 1% agarose gel and analyzed. The arrowheads denote positive signals in 28S and 18S rRNA. The arrow probably denotes the 28S rRNA-derived degraded transcript produced during the sample preparation.
Fig 5.
Detection of stress-induced tRNA-derived fragments by immuno-northern blotting.
(A) The image of stress-induced tRNA cleavage. tRNAs are cleaved under conditions of cell stress, and consequently, tRNA-derived fragments such as the 5’-side and 3’-side fragments appear. The modification of m1A is usually contained in the 3’ side of the tRNA structure. (B) Total RNA (0.75 μg) was isolated from HK-2 cells treated with sodium arsenite (500 μM) for the indicated periods and analyzed by SYBR staining and INB with anti-m1A antibody using acrylamide gel separation. Arsenite-stress induced tRNA cleavage, and the resultant tRNA-derived fragments were detected by SYBR staining and firmly detected by the INB with anti-m1A antibody.
Fig 6.
Specificity and sensitivity of immuno-northern blotting.
(A) The sequences of synthesized 15-mer ssRNA containing either m6A or A at the center position. (B) SYBR staining and INB with anti-m6A antibody. Serially diluted m6A- and A-containing 15-mer ssRNA (10, 4, 1.6, 0.64, and 0.25 pmol) were analyzed. (C) Relative band intensities by INB of m6A-containing ssRNA. Densitometric analysis was performed to quantitate the band intensities. Data were expressed relative to the band intensity of each 10 pmol sample, which was taken as 1.0 arbitrary units (AU), N = 3. Values are shown as the mean ± SD.
Fig 7.
Detection of m6A-demethylation by immuno-northern blotting.
(A) Demethylation of m6A to A in RNA by FTO and ALKBH5. (B) m6A-containig 15 mer-ssRNA was treated with or without FTO and analyzed by INB with anti-m6A antibody and SYBR. The bottom graph shows a densitometric quantification of the band intensity. Data were expressed relative to the mean value of the FTO (-) samples, which was taken as 1.0 AU, N = 3. (C) m6A-containig 15 mer-ssRNA was treated with or without ALKBH5 and analyzed by INB and SYBR. A representative image is shown. (D) LC-MS/MS analysis of FTO-mediated demethylation. After the FTO treatment, m6A-ssRNA was digested and subsequently analyzed by LC-MS/MS. Chromatograms of the m6A peak of digested nucleosides are shown. The right graph shows a quantification of the relative area ratio of m6A/A. Data were expressed relative to the mean value of the FTO (-) samples, which was taken as 1.0. (E) Knockdown of FTO and ALKBH5 in HeLa cells. The knockdown efficiency was measured by quantitative PCR. Relative abundance of each transcript was normalized by GAPDH. Data were expressed relative to the mean value of the control, which was taken as 1.0, N = 3. (F) Evaluation of the m6A modification in RNAs in FTO- and ALKBH5-knockdown HeLa cells. The mRNA (100 ng) and total RNA (1.0 μg) were analyzed by INB with anti-m6A antibody using acrylamide gel separation. Arrowheads denote the anti-m6A positive signals. SYBR staining is shown for the loading control. Values are shown as the mean ± SD. *P < 0.05.