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. Author manuscript; available in PMC: 2020 Mar 30.
Published in final edited form as: Nat Genet. 2019 Sep 30;51(10):1518–1529. doi: 10.1038/s41588-019-0502-z

Figure 1. NPM1 regulates 2’-O-methylation through snoRNA binding.

Figure 1.

a, RNA binding motifs of NPM1 identified by NPM1 HITS-CLIP. b, Functional annotation of the RNA species identified in NPM1 HITS-CLIP. c, Levels of specific 2’-O-Me residues (x-axis) mediated by C/D box snoRNAs in HITS-CLIP. Fold change was calculated Npm1−/− relative to Npm1+/+ MEFs. Data are presented as mean ± SD of n = 3 independent experiments and compared to levels of Ctrl modification. Ctrl is U1804 that is mediated by snoRD20, which was not identified in HITS-CLIP. d, FBL-IP was performed using Npm1+/+ and Npm1−/− MEFs. Snord enrichment was calculated relative to total input and fold enrichment in Npm1+/+ MEFs and compared to levels of enrichment of SnordCtrl, snoRD13 which was not identified to interact with NPM1 by HITS-CLIP. Bars present mean ± SD of n = 4 independent experiments. e, IP of either NPM1 (upper panel) or FBL (lower panel) was performed using nuclear extracts of Npm1+/+ and Npm1−/− MEFs. Shown is representative image out of 4 independent experiments. f, KEGG pathway analysis of Npm1+/− polysome-microarray analysis relative to Npm1+/+. Statistical significance was determined by gene set enrichment analysis. g, Cumulative distribution of minimum free energy (MFE) of 5’UTRs of polysome-depleted and -enriched transcripts, normalized to 5’UTR length. h, Diagram of the dual luciferase reporter testing for both cap-dependent and IRES-dependent translation. i, Npm1+/+, Npm1+/− and Npm1−/− MEFs were transfected with the Cap/IRES luciferase reporter (diagram in h). Bars represent SD of n = 6 independent experiments. j, Western blot of the IRES translated genes p27 and XIAP, in Npm1+/+ and Npm1−/− MEFs. RPL22 served as a loading control. Shown is representative data out of 6 independent experiments. k, 5’UTRs of Cdkn1b and Xiap lead to reduced translation of the luciferase reporter in Npm1−/− compared to Npm1+/+ MEFs. IRES versus Cap translation ratio was calculated relative to the activity in Npm1+/+. Data are presented as mean ± SD of 3 independent experiments, and compared to levels in Npm1+/+ MEFs. l, Transcript enrichment in Npm1−/− activated ribosomes (pS6-IP) was calculated relative to the enrichment in Npm1+/+ MEFs. Data are presented as mean ± SD of 4 independent experiments. m, IRES activity was calculated relative to the activity in Npm1+/+ MEFs. Data are presented as mean ± SD of 3 independent experiments. n, Analysis of specific 2’-O-Me in Clinical Remission (CR, n = 14) and NPMc+ AML patients (NPMc+, n = 16). Error bars represent SEM. Statistical significance was calculated by Mann-Whitney analysis. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed Student’s t test. Uncropped blot images are presented in Supplementary Figure 11.