Journal: Nucleic Acids Research
Article Title: N 2 -methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing
doi: 10.1093/nar/gkad487
Figure Lengend Snippet: tRNA m 2 G modifications are important for proliferation of HCT116 colorectal cancer cells and optimal translation. ( A ) Schematic representation of TRMT11 and THUMPD3 showing their domain organization. The predicted amino acid boundaries of the different domains are shown above each diagram. The MTase domains are colored in orange. The strongly conserved signature found in the MTase domains, which is assumed to coordinate the substrate into the MTase active site, is indicated. ( B ) Western blot analyses of HCT116 TRMT11 (top left panel), THUMPD3 (top right panel) or double (lower panel) knockout cell lines. Proteins of interest were detected by western blot using the indicated antibodies. Three biologically independent experiments were performed and a representative image is shown. ( C ) Quantification by LC–HRMS of m 2 G (left black axis) and m 2,2 G (right red axis) levels in total tRNAs purified from the indicated cell lines. Mean values calculated from five replicates are shown and error bars represent standard deviation. ( D ) Growth analysis of WT, TRMT11, THUMPD3 or TRMT11/THUMPD3 KO HCT116 cell lines. Cell numbers were monitored every 24 h for 6 days. Three biologically independent experiments were performed and error bars represent standard deviations. ( E ) Total RNAs extracted from the indicated cell lines were separated by native polyacrylamide gel electrophoresis and analyzed by northern blotting using probes hybridizing to the tRNAs indicated on the left. The presence and position of m 2 G in the detected tRNAs is noted on the right. Three biologically independent experiments were performed and a representative image is shown. ( F ) Total RNAs were extracted from the indicated cell lines under acidic conditions. A sample subjected to alkaline treatment served as a deacylated control. Samples were separated by denaturing polyacrylamide gel electrophoresis under acidic conditions before analysis by northern blotting using probes hybridizing to the tRNAs indicated on the right. The presence and position of m 2 G in the detected tRNAs is also given. Three biologically independent experiments were performed and a representative image is shown. ( G ) Polysome profile analyses using the indicated cell lines were performed. For each cell line, the mean areas of each of the ribosomal complexes (small subunit – 40S, large subunit – 60S, monosomes – 80S and polysomes) observed in the polysome profile analyses for each cell line is shown. Non-statistically significant differences are not indicated for the sake of clarity. Five biologically independent experiments were performed and error bars represent standard deviations. ( H ) Effect of TRMT11 and/or THUMPD3 depletion on overall protein synthesis. For TRMT11 KO2 and TRMT11/THUMPD3 KO1 cell lines, the GAPDH signal was used to normalize the data, whereas for the THUMPD3 KO1 cell line, we used the alpha-tubulin signal as GAPDH levels are affected in this cell line. Three biologically independent experiments were performed and error bars represent standard deviations.
Article Snippet: As presence of modified nucleotides can influence tRNA stability or folding ( , ), tRNA levels in the KO cell lines were first comprehensively determined using the nrStarTM human/mouse tRNA PCR array service from Arraystar Inc. (Rockville, MD 20850, USA; and ).
Techniques: Western Blot, Knock-Out, Purification, Standard Deviation, Polyacrylamide Gel Electrophoresis, Northern Blot, Control
