Fig 2: Modification of the nascent proteome by TIAR or FMRP silencingK562 cells transduced with shNEG, shTIAR or shFMRP (shRBP) were cultured for 72 h in N or CO before incorporation for 4 h of Met analog (L-HPG) and ‘heavy’ or ’medium’ Lys or Arg for QuaNCAT-MS. Results from 3 independent experiments.(A) Comparison of proteins upregulated (fold change log2>0.6, UP) or downregulated (log2<-0.6, DOWN) in shTIAR or shFMRP versus shNEG, in N or CO conditions. Total number of proteins in the group is in gray italic, and overlapping between the compared groups - in the Venns’ graphs.(B) Proteins exclusively regulated by shTIAR or shFMRP in CO condition analyzed for functional enrichment by string.db; proteins marked according to the legend below the graph; terms over-represented with Benj.Hoch. FDR=0.05.(C and D) Protein level in total cellular extract analyzed by Western blotting.

Fig 3: Microenvironment-induced changes in the profile of proteins present in cytoplasmic complexes of TIAR and FMRP proteinsMass spectrometry analysis combined with tandem mass tagging (TMT-MS) was used to study the proteome of complexes immunoprecipitated (IP) from cytoplasm of K562 cells cultured in N or CO setup for 72 h. Profile of proteins isolated by IP with antibody of the same isotype (iso IgG) as the antibody used for IP of TIAR and FMRP (specific IgG) used as a background control. In total 6 isolations from each condition and IP were combined for isobaric labeling and analyzed by mass spectrometry in a single run.(A) Scheme of experiment.(B and C) Western blotting analysis of IP prepared with IgG isotype (iso) and FMRP (B) or TIAR (C) from K562 untreated (-) or treated (+) with RNase A for 1 h; input - signal in the cytoplasmic fraction used for IP. Representative images presented, n = 3.(D and E) Analysis of proteins identified in TMT-MS. Comparison of proteins with average ratio RBP/ISO =2 from 3 experiments (1–3 following the sample name) presented as (D) heatmap; legend on the right – in white ratio =1.5; in dark red ratio =10; (E) principal component analysis.(F–H) Analysis of proteins with significant (p<0.05) change of ratio RBP/ISO =1.5.(F) Number of ribosomal proteins (RP) identified in the samples (both N and CO).(G) Assessment of enrichment in the samples in proteins annotated KEGG:Ribosome03010.(H) Comparison of presence of small (RPS) and large (RPL) ribosomal subunits proteins in the IP complexes of TIAR and FMRP from N and CO conditions – fold change (FC) values of RBP/ISO are presented on the heatmap, n = 3; 1–3 – single experiments, AVG - mean value of n = 3; legend in left up corner – in white FC =1.5, in dark red the highest FC >2.5; both - proteins present in complex with TIAR (T) and FMRP (F).(I and J) Impact of CO versus N on RP level in FMRP (I) or TIAR (J) IP complexes; selected RPs in red.

Fig 4: Knock-down of FMRP or TIAR have impact on the translation process(A and B) Total cellular lysates of K562 cells transduced with 5 different lentiviral particles bearing different shRNA (C1-C5) were checked for TIAR (A) and FMRP (B) protein knock-down by Western blotting and compared with parental (Par) and transduced with control virus (shNEG) cells; a black triangle - clones used further.(C) Level of AHA-AlexaFluor 488 labeled proteins in K562 cells from N, H or CO conditions measured by flow cytometry; 100 nM homoharringtonine (HHT) in N for control.(D) IRES- (F) and cap- (R) dependent translation (left panel) and ratio IRES/cap (right panel) tested by dual-luciferase assay. (C and D) Fold change to N conditions, mean values ± ME, n = 4. Student’s ttest to compare cells in N with H or N with CO (*) and one-way ANOVA to compare shTIAR to shNEG and shFMRP in H or CO (#); *p<0.05, **p<0.005, ****p<0.0005, ns – not significant.(E) Profile of ribosomal mono-/polyribosomes separated in sucrose linear gradient 10–50%; absorbance monitored at ? = 254 nm, normalized to protein level in the input.(F) Distribution of ribosomal proteins in the fractions (numbered 1–8) analyzed by Western blotting - densitometry of results by ImageJ as a % of a sum signal intensity of bands in all 8 fractions on a blot; mean values ± ME, n = 3; Student’s t test to compare cells shNEG to shTIAR; *p<0.05.