Extended Data Fig. 2: HNRNPC binding impacts the translation and alternative polyadenylation of its targets.
From: An mRNA processing pathway suppresses metastasis by governing translational control from the nucleus
(a) Cumulative density plot of translation efficiency ratios (TER) comparing sgHNRNPC to sgControl MDA-MB-231 cells, for HNRNPC 3′ UTR target and non-target mRNAs. Median difference (∆M) and p-value (calculated using two-tailed Mann-Whitney U-test) are shown. (b) Two-dimensional heatmap showing significant logTER correlation of translationally repressed mRNAs in MDA-LM2 and HNRNPC knockdown (sgHNRNPC) cells. For comparison, the Spearman correlation coefficient and the associated p-value are shown across all genes. (c) TXNRD1 and PGK1 mRNA levels in MDA-MB-231 and MDA-LM2, as well as sgControl and sgHNRNPC-expressing cells, as determined by RNA-seq. (d) Western blot analysis of TXNRD1 and PGK1 proteins in MDA-MB-231 and MDA-LM2, as well as sgControl and sgHNRNPC-expressing cells. Relative protein quantity normalized to GAPDH is indicated below, along SE. The western blot was performed once in biological triplicates to confirm the TMT-MS results. (e) Cumulative density plot of alternative polyadenylation ratios (logAPAR) comparing MDA-LM2 to MDA-MB-231 cells, for HNRNPC 3′ UTR target and non-target mRNAs; statistics as in (a). (f) Cumulative density plot of logAPAR comparing sgHNRNPC to sgControl cells, for HNRNPC 3′ UTR target and non-target mRNAs; statistics as in (a). (g) Two-dimensional heatmap showing significant logAPAR correlation of proximal to distal poly(A) site switch in MDA-LM2 and HNRNPC knockdown (sgHNRNPC) cells; statistics as in (b). (h) Bidirectional promoter reporter schematics, used for massively parallel reporter assays (MPRA). (i) BFP/mCherry ratio of reporter-expressing MDA-MB-231 cells, transfected with control or HNRNPC-targeting siRNAs, as detected by flow cytometry. (j) Box plot illustrating the relative reporter logAPAR, comparing transcripts with HNRNPC-binding sites versus matched scrambled controls, in control and HNRNPC KD cells. n = 2 biological replicates. p-value calculated using one-tailed Wilcoxon signed-rank test. Box center reports the median value, the boundaries - the quartiles, and the whiskers - the 10 and 90 percentiles. (k) Box plot illustrating the relative reporter logAPAR, comparing transcripts with HNRNPC-binding sites versus matched scrambled controls, in control and HNRNPC KD cells, stratified by reporter protein expression (25% high versus 25% low BFP/mCherry ratio, as determined and sorted by flow cytometry). n = 2 biological replicates. p-value calculated using one-tailed t-test. Box plot characteristics as in (j).
