Extended Data Fig. 1: Validation and characterization of selective MYC translation screen candidates.
From: Functional screen identifies RBM42 as a mediator of oncogenic mRNA translation specificity
(a) Volcano plot showing positive control known activators and negative control genes of MYC translation from Panc-1 CRISPRi reporter screen. eIF5A was previously identified and validated as the sole hit in a MYC translational repressor screen25. Mann–Whitney p-value, two-sided. (b) Gene ontology molecular function enrichment for the 309 candidate repressors of MYC translation in Panc-1 cells. Gene ratio is the fraction of genes identified within the GO term. Fisher’s Exact Test with Bonferroni correction. (c) mRNA expression of UBAP2L, METTL3 and YTHDF2 in healthy pancreas (GTex) versus pancreatic adenocarcinoma (PDAC)62; Welch’s t-test, two-tailed. (d) Protein expression of UBAP2L, METTL3 and YTHDF2 normal versus tumour from CPTAC dataset; two-sided t-test41. Normal/primary tumour UBAP2L maximum: 0.81/2.133, upper quartile: −0.314/0.63, median: −0.678/0.009, lower quartile: −1.052/−0.805 and minimum: −1.933/−2.799. Normal/primary tumour METTL3 maximum: 1.804/1.932, upper quartile: 0.937/0.503, median: 0.308/−0.71, lower quartile: −0.454/−0.71 and minimum: −2.687/−2.486. Normal/primary tumour YTHDF2 maximum: 0.85/2.05, upper quartile: 0.014/0.547, median: −0.47/0.037, lower quartile: −0.912/−0.761 and minimum: −2.097/−2.015. (e) Single guide validation with 2 independent sgRNAs in the Panc-1 MYC translational reporter cell line used for the CRISPRi screen at day 5 doxycycline treatment, positive controls EIF4A1 and EIF4H. sgEIF4A1 n = 18; sgEIF4H, sgRBM42, sgUBAP2L, n = 4; sgMETTL3 and sgYTHDF2 n = 6. (f) qPCR RNA quantification of single guide validation experiments. sgEIF4A1 n = 12; sgEIF4H and sgRBM42 n = 4; sgUBAP2L, sgMETTL3 and sgYTHDF2 n = 2. (g) Single guide validation of negative control genes with 2 independent sgRNAs in the Panc-1 MYC translational reporter cells. sgEIF4A1 n = 18, sgEIF3E and sgMSI1 n = 6, sgRBM23-1 n = 8, sgRBM23-2 and ssgSF3B1 n = 6. (h) qPCR RNA quantification of negative control gene experiments. sgEIF4A1 n = 9, sgEIF3E and sgMSI1 n = 3, sgRBM23-1 n = 4, sgRBM23-2 and sgSF3B1 n = 3. (i) Quantification of RNA from UBAP2L, METTL3 and YTHDF2 knockdown experiments in Panc-1 cells. shCNTL n = 7, shUBAP2L n = 6, shMETTL3 and shYTHDF2 n = 3. (j) qPCR analysis of MYC mRNA from 10–50% sucrose gradient fractionation of control or YTHDF2 knockdown in Panc-1 cells, n = 3. (k) Total RNA quantification from UBAP2L, METTL3 and YTHDF2 knockdown polysome experiments, n = 3. (l) qPCR analysis of control genes, ACTB or B2M, from 10–50% sucrose gradient fractionation of control or YTHDF2 knockdown in Panc-1 cells, n = 3. (m) Correlation of UBAP2L and Myc protein abundance from human pancreatic adenocarcinoma specimens41, n = 17. All graphs mean ± SEM. Total RNA quantification in i,k one-way ANOVA, uncorrected Fisher’s LSD test relative to own shCNTL. Polysome distribution in j two-way ANOVA, uncorrected Fisher’s LSD test. Extended data associated with Fig. 1.
