Fig 2: Compartment-specific RBP capture by eRIC is highly specific and sensitive. (A, B) Inputs and 10% of RNase eluates from eRIC captures were separated by SDS-PAGE and the proteins were visualized by silver staining (A) and western blotting (B). 250 ng and 0.05% of inputs were loaded onto the gels for silver staining and western blotting, respectively. (B) The western blots were immunoblotted against the known RBPs TIA-1 and ELAVL1, or against the non-RNA-binding proteins Histone H4 and α-tubulin as negative controls. The observed upwards shift in gel mobility of TIA-1 and ELAVL1 between the inputs and eluates of the UV-crosslinked samples is likely due to the presence of small cross-linked RNA fragments in the eluates. (C–H) captured RNA species were analyzed by RT-qPCR. The relative abundance of mRNA (C, D), rRNA (E, F), gDNA (G), and pre-mRNA (H) in cytoplasmic (Cyto) and nuclear (Nuc) inputs and eluates, from untreated and arsenite-treated (+ars) cells were quantified. The data represent the mean with standard deviation from three biological replicates. For analysis of captured pre-mRNA and gDNA samples where reverse transcriptase (-RT) was omitted from the cDNA synthesis was included to evaluate the presence of a gDNA-derived template. Note that mRNA is effectively captured by eRIC while rRNA is strongly selected against compared to inputs. Also gDNA contamination is low as observed by minor amplification in –RT samples. Further, the pre-mRNA capture is restricted to the nuclear fractions and eRIC efficiently discriminates against gDNA capture as observed by only minor amplification in –RT eluates (note the log10 scale in the Y-axis). The horizontal lines marked (*) above the columns indicate the compared groups. Long vertical ticks indicate the control-samples and short ticks indicate P< 0.05 (unpaired two-tailed t-test).

Fig 3: TIA1 antibody screening by immunofluorescence.HAP1 WT and TIA1 KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated TIA1 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given to antibodies ab140595**, A6237, NBP2-67203**, and GTX33545, which were titrated to their respective concentrations found bellow, as the signals were too weak when following the suppliers' recommendations. When the concentrations were not indicated by the supplier, which was the case for antibodies ab263945**, ARP40979, ARP40981, and NBP3-13791** we tested antibodies at 1/300, 1/500, 1/500 and 1/200, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab140595** at 1/600; ab263945** at 1/300; A6237 at 1/1000; ARP40979 at 1/500; ARP40981 at 1/500; NBP2-53336* at 1/100; NBP2-67203** at 1/1000; NBP3-13791** at 1/200; 86050** at 1/100; GTX33545 at 1/800; MA5-26474* at 1/1000; and MA5-32615** at 1/100. Bars = 10 μm. *=monoclonal antibody, **=recombinant antibody.

Fig 4: TIA1 antibody screening by Western Blot.Lysates of HAP1 (WT and TIA1 KO) were prepared and 50 μg of protein were processed for Western Blot with the indicated TIA1 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Antibody dilution used: ab140595** at 1/5000; ab263945** at 1/1000; A6237 at 1/1000; ARP40979 at 1/500; ARP40981 at 1/200; NBP2-53336* at 1/1000; NBP2-67203** at 1/1000; NBP3-13791** at 1/500; 86050** at 1/1000; GTX33545 at 1/500; MA5-26474* at 1/2000; and MA5-32615** at 1/500. Predicted band size: 43 kDa. *=monoclonal antibody, **=recombinant antibody.

Fig 5: TIA1 promotes ULK1 protein expression and autophagy during nutrient starvation(A) Loss of TIA1 reduces ULK1 protein expression and autophagy. Calu-1 cells transfected with either siRNA (control) or siRNA specific to TIA1 were subjected to serum starvation for the indicated times with or without treatment with bafilomycin A1. Cells were harvested after the indicated times and protein levels of ULK1, TIA1, and LC3 determined by immunoblotting. ACTB was used as a loading control.(B) Quantification of ULK1 protein levels in (A). Data represent three independent biological replicates, showing mean ± SD. ****p < 0.001.(C) TIA1 knockdown does not affect ULK1 transcription. Total RNA was extracted from cells harvested under the conditions indicated in (A), and ULK1 mRNA abundance was determined by RT-qPCR. GAPDH was used as a control. Data represent three independent biological replicates.(D) Quantification of LC3-II levels in (A). Data represent three independent biological replicates, showing mean ± SD. two-way ANOVA was used to determine statistical significance. ****p < 0.01, ***p < 0.05.(E) TIA1 binds to the 3′ UTR of ULK1. RNA immunoprecipitation was performed using TIA1-specific antibody, and the mRNA bound to the protein was quantified by RT-qPCR using two primers spanning different regions in the 5′ UTR and 3′ UTR. Data represent three independent biological replicates, showing mean ± SD. One-way ANOVA was performed to determine statistical significance. **p = 0.0013, *p = 0.0358.