Supplementary Fig. 2: NPM1-deletion affects specific modes of translation and does not affect global translation.
From: Germline NPM1 mutations lead to altered rRNA 2′-O-methylation and cause dyskeratosis congenita
a, 35S-Methionine incorporation assay of Npm1+/+ and Npm1−/− MEFs showed no differences on global protein synthesis. Quantity of labeled proteins was analyzed by radiography (RG). n=3 independent experiments. b, HPG metabolic labeling of Npm1+/+ and Npm1−/− MEFs showed no difference in global protein synthesis. Detection of HPG labeling was by azide AlexaFlour-647 and flow cytometry. n=3 independent experiments. c, Flow cytometry analysis of forward scatter and side scatter of Npm1+/+ and Npm1−/− MEFs. n=3 independent experiments. d, Analysis of total RNA content of Npm1+/+ and Npm1−/− MEFs. n=3 independent experiments. e, Northen blot of rRNA processing was performed using Npm1+/+ and Npm1−/− cells. n=3 independent experiments. f, WB analysis of mTOR downstream effectors in Npm1+/+ and Npm1−/− MEFs. HSP90 served as a loading control. n=3 independent experiments. g, Top panel: gene set enrichment (GSEA) analysis using the full set of GFP-positive genes showed statistically significant negative enrichment of putative IRES genes, NES = -1.359, p-value = 1.167e-4. Bottom panel: GSEA analysis of the restricted list of 583 IRES genes identified in Weingarten-Gabbay et al. and found a negative trend of IRES genes associated with Npm1+/- polysome-depleted transcripts, NES = -1.049, p-value = 0.31. h, WB of NPM1 in Npm1+/+, Npm1+/- and Npm1−/− MEFs. Shown is a representative blot of n=3 independent experiments. i, SnoRNA abundance in Npm1+/+ and Npm1+/- MEFs. Data are presented as mean±SD, n=4 independent experiments. j, Levels of specific 2′-O-Me residues (x-axis) mediated by only C/D box snoRNAs identified to interact with NPM1 by HITS-CLIP in Npm1+/- MEFs. Data are presented as mean±SD (n=3 independent experiments) of fold change of 2′-O-Me in Npm1+/- MEFs relative to Npm1+/+. Ctrl residue is the U1804 2′-O-Me modification on the 18S rRNA that is mediated by snoRD20, which was not identified to interact with NPM1 by HITS-CLIP. k, qPCR analysis of Cdkn1b (p27 transcript) and Xiap levels in Npm1−/− MEFs relative to Npm1+/+. Data are presented as mean±SD, n=3 independent experiments. l-m, Ribosome fractionation was performed using Npm1+/+ and Npm1−/− MEFs. ActinB, Gapdh and β2-microglobulin served as cap-translated controls (l). Cdk1nb, Xiap, Vegf and Fgf2 are IRES-translated genes presented in m. mRNA enrichment in polysome fractions is presented as percent from total RNA±SD. n=3 independent experiments. 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 Fig. 11.
