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

Fig 3: The easyCLIP protocol.a easyCLIP schematic. b Diagrams of the 5′ and 3′ adapters with IR680 dye (LI-COR) and IR800-CW dye, respectively. c A nitrocellulose membrane representing the gel in panel (a), in which CLIP libraries produced for the protein hnRNP C are visualized by the dyes on the adapters, the 5′ in red and the 3′ in green. The size of uncross-linked hnRNP C noted at left. “Larger complexes” contain ligated adapter and hnRNP C protein; they likely reflect the presence of additional proteins, but their exact nature is unknown. The experiment was performed three times with similar results. d Spearman’ ρ values for reads per gene from replicates of 11 RBPs, excluding RPL5 as it has only one definitive target RNA (5S rRNA). Endogenous hnRNP C, RBFOX2, and FBL were immunopurified, HA-PCBP1 was stably integrated, and the others were transiently expressed as HA-tagged forms. PCBP1 was expressed in HCT116 cells and the others in 293Ts. e The most significant motif identified de novo by HOMER for the top mRNA peaks. Motif enrichment significance computed by HOMER (hypergeometric test with p value corrected for multiple testing). f The most significant motif identified de novo by HOMER for all peaks significantly enriched over randomly chosen non-RBPs. g Fraction of easyCLIP reads of the indicated RBP mapping to RNAs of the given ENSEMBL biotype. h Fraction of reads mapping to rRNA. i Fraction of reads across all RNA types (left) or across mRNA (right) that map to introns or exons. j Number of significantly enriched (FDR < 10−4) categories of RNAs, relative to randomly selected non-RBP controls. “Mill. reads” denotes the number of uniquely mapping read pairs. k Fraction of FBL cross-link locations normalized to their average length across all C/D-box snoRNAs; one dot represents one nucleotide in the normalized snoRNA.