Fig 1.
Characterization of the global transcriptomes of RBP-expressing FT293 cells by massive sequencing.
(A) Expression patterns of GFP-tagged proteins. Fluorescence images together with corresponding phase contrast photographs (merge) are shown for 3 days. Scale bars represent 20 μm. (B) Working pipeline of the RNA-seq analysis. Workflow followed to analyze the datasets from massive-scale RNA sequencing. The legend identified as G, 1, R, and H indicates GFP, TIA1b, TIARb, and HuR samples, respectively.
Fig 2.
Gene Ontology (GO) analysis of differentially expressed genes (DEGs) in GFP-, GFP-TIA1b-, and GFP-TIARb-expressing FT293 cells.
(A-H) Top categories of biological processes associated with DEGs. (A and E) MA plot representations of the distributions of up- (spots in red) and down- (spots in green) expressed genes (-1 ≥ log fold change ≤ 1; FDR < 0.05) in the corresponding combinations indicated in A and E panels, respectively. (B-D and F-H) Histograms of the distributions of up- and down-regulated genes (B and F), only up-regulated genes (red in C and G) and only down-regulated genes (green in D and H) in the corresponding combinations using the GO PANTHER database (P < 0.05). (I) Venn diagram displaying numbers and GO enriched categories of DEGs in TIA1- and TIAR-expressing FT293 cells compared with control (GFP-expressing FT293 cells).
Fig 3.
Gene Ontology (GO) analysis of differentially expressed genes (DEGs) in GFP- and GFP-HuR-expressing FT293 cells.
(A-D) Top categories of biological processes associated with DEGs. (A) MA plot representations of the distributions of up- (spots in red) and down- (spots in green) expressed genes (-1 ≥ log fold change ≤ 1; FDR < 0.05) in the corresponding combinations indicated in A panel. (B-D) Histograms of the distributions of up- and down-regulated genes (B), only up-regulated genes (red in C) and only down-regulated genes (green in D) in the corresponding combinations using the GO PANTHER database (P < 0.05). (E and F) Venn diagrams displaying numbers and GO enriched categories of DEGs in the comparisons HuR vs GFP against TIA1 vs GFP (E) and HuR vs GFP against TIAR vs GFP (F) combinations.
Fig 4.
Gene Ontology (GO) analysis of differential splicing events (DSEs) in RBP-expressing FT293 cells.
(A-C) Top categories of biological processes associated with DSEs using the GO PANTHER database (P < 0.05). Histograms of the distributions of DSEs in the pairs TIA1 vs GFP (A), TIAR vs GFP (B) and HuR vs GFP (C) are shown. (D) Scheme and legends of differential alternative splicing events (ASEs) detected by multivariate analysis of transcript splicing (MATS) taken from Xing lab (http://rnaseq-mats.sourceforge.net). (E and F) Bar plots represent the number of each type of alternative splicing event detected using MATS. (E) JC: total number of events detected using junction counts only. JC+ROT: total number of events detected using both junction counts and reads on targets. (F) JC_sig and JC+ROT_sig represent number of significant events in the above categories, respectively.
Fig 5.
Short-term effect of ectopic RBP expression on rates of cell proliferation, relative translation, subcellular localization, and cell cycle.
(A) Rates of cell proliferation. Cells were grown in culture medium containing glucose (10 mM) and counted at 24–72 h. Values indicate means ± SEM (n = 3; *P < 0.05; **P < 0.01). (B) Rates of global translation. Translational rates of total proteins in above cells for 24–72 h were determined in the presence of 35S-methionine/-cysteine (35S-Met/Cys). The relative translational rates were estimated as ratio 35S-Met/Cys label versus Coomassie Blue reagent staining. The values are indicated as percentage relative to control sample (GFP). The legend identified as G, 1, R, and H indicates GFP, TIA1b, TIARb, and HuR samples, respectively. (C) Rates of subcellular localization. The above cell lines were cultured for 48 h, stained with Mitotracker and visualized by confocal microscopy. GFP-tagged proteins and mitochondria are visualized in green and red, respectively. Scale bars represent 10 μm. (D) Analysis of cell cycle phases by flow cytometry. The percentage of indicated cells was quantified in every cell cycle phase for each experimental condition indicated. Values represent means ± SEM (n = 3; *P < 0.05; **P < 0.01). (E) The expression of TIA1b and TIARb in FT293 cells drives cell cycle arrest at the G1/S phase. Cell lines were synchronized at G1/S by hydroxyurea blockage for 24 h, released and quantified at 16 h post-release (h.p.r.).
Fig 6.
Analysis of autophagy, mitophagy, and apoptosis in RBP-expressing cells for 24–72 hours.
(A) Autophagy analysis. Cell lines were transfected with GFP-LC3B-RFP plasmid, visualized by confocal microscopy and quantified. Histograms represent the sum of autophagosomes and autolysosomes estimated as yellow and free red dots per cell, respectively. The represented values were normalized and expressed as percentage relative to the GFP control. (B) Mitophagy analysis using mitochondrial Keima probe. Cell lines were transfected with the mt-Keima-expressing plasmid, visualized by confocal microscopy and counted. Histograms show normalized red/green mt-Keima signals. The represented values were expressed as a percentage relative to the GFP control. (C) Rates of cell death by apoptosis for 3 days. Quantification of apoptosis rates was carried out by flow cytometry. All the represented values are means ± SEM (n = 3; *P < 0.05).
Fig 7.
Regulatory crosstalk between the expression of TIA proteins and EIF2AK2-mediated cell quiescence.
(A and B) Validation and quantification of relative expression levels of RNAs and proteins by qPCR (A) and western blot (B) analysis. Protein extracts and cytoplasmic RNAs were isolated from GFP-, TIA1b-, TIARb-, and HuR-expressing FT293 cells and analyzed by qPCR (A) and western blotting (B). The represented values were normalized and expressed relative to the GFP control (A). The values are means ± SEM (n = 2–3; *P < 0.05; **P < 0.01). For western blot analysis were used the antibodies indicated. (C) Effect of the disruption of mouse TIA1, TIAR and EIF2AK2 genes on TIA1, TIAR and EIF2AK2 expression in murine embryonic fibroblasts (MEFs). Protein extracts from normal (WT MEF) or knocked out for TIA1 (KO1 MEF), TIAR (KOR MEF) or EIF2AK2 (KOE MEF) were purified and processed for western blot analysis using the antibodies indicated. (D) EIF2AK2 knockdown using lentiviral particles in TIARb-expressing FT293 cells. Western blot analysis of protein extracts of TIARb-expressing FT293 cells (without tetracycline means in the absence of TIARb expression) stably transfected with shRNAs against control (C) and EIF2AK2 (E) using a lentiviral transfection. The blot was probed with specific antibodies against EIF2AK2, HuR/ELAVL1 and TUBA proteins. (E) TIARb-expressing FT293 cells transfected with shRNAs against control (C) and EIF2AK2 (E) were seeded and grown for 3 days in the absence (-) and presence (+) of tetracycline. Above cells were monitored by flow cytometry after propidium iodide staining. The data are means ± SEM of the three independent experiments (n = 3; *P < 0.05; **P < 0.01). (B-D) Molecular weight markers for protein (kDa) and the identities of protein bands are indicated on the left and right by arrowheads, respectively.
Fig 8.
A working model summarizing the molecular and cellular events associated with the ectopic short-term expression of TIA proteins.