Fig. 6: TFIP11 but not DHX15 is required for U6 snRNA 2’-O-methylation. | Nature Communications

Fig. 6: TFIP11 but not DHX15 is required for U6 snRNA 2’-O-methylation.

From: DHX15-independent roles for TFIP11 in U6 snRNA modification, U4/U6.U5 tri-snRNP assembly and pre-mRNA splicing fidelity

Fig. 6

a, b Identification of 2′-O-methylation on U snRNAs in HCT116 cells transfected for 72 h with control siRNA (siCtr), one of two different siRNAs against TFIP11 (siTFIP11 #1 and siTFIP11 #2) or one of two different siRNAs against DHX15 (siDHX15 #1 and siDHX15 #2). The heatmap displays the proportion of methylation for each modified position (column). Modified positions are highlighted as follows: asterisk = a position particularly sensitive to TFIP11 knockdown; red = hypomodification. b, c Protein extracts were analyzed by western blotting with antibodies against TFIP11 (b), DHX15 (c), and β-actin (loading control). d Identification of 2′-O-methylation on U snRNAs in HCT116 cells transfected for 72 h with control siRNA (siCtr) or one of two different siRNAs against coilin (siCoilin #1 and siCoilin #2). The modified positions are highlighted as follows: asterisk = a position particularly sensitive to coilin knockdown; red = hypomodification. d Protein extracts were analyzed by western blotting with antibodies against coilin and β-actin (loading control). f Immunoprecipitation (IP) of endogenous FBL from HeLa cell extracts followed by western blotting (WB) for the indicated proteins. g Western blotting for the indicated proteins after RNA IP (RIP) shows the IP of FBL in HeLa cells mock-transfected (No siRNA) or transfected with control siRNA (siCtr) or one of two different siRNAs against TFIP11 (siTFIP11 #1 and siTFIP11 #2). h–k Immunopurification of RNA-FBL complexes and RT-qPCR for U6 snRNA (overall p = 0.022, p = 0.032 for TFIP11#1, p = 0.038 for TFIP11#2, n = 4 independent experiments) (h) and its guide snoRNAs: SNORD7 (overall p = 0.019, p = 0.035 for siTFIP11#1, p = 0.029 for siTFIP11#2, n = 4 independent experiments) (i), SNORD67 (overall p = 0.019, p = 0.013 for siTFIP11#1, p = 0.011 for siTFIP11#2, n = 4 independent experiments) (j) and SNORD94 (overall p = 0.036, p = 0.074 for siTFIP11#1, p = 0.04 for siTFIP11#2, n = 4 independent experiments) (k) in HeLa cells transfected as in g. β-actin mRNA in IP beads served as an internal qPCR normalization reference. p-values were calculated using repeated measures ANOVA with post-hoc two-tailed paired t-tests. Only p-values < 0.05 (relative to siCtr) are displayed. Box limits = 25th to 75th percentiles; line = median; whiskers = min to max. Source data are provided as a Source Data file.

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