The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression

Abstract

TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5' as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR and stress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.

Keywords: T-cell restricted intracellular antigen-1; alternative splicing; apoptosis; cell cycle; eukaryotic translation initiation factor 2 alpha kinase 2; integrated stress response; interferon-inducible double-stranded RNA-dependent protein kinase activator A.

Figure 1. DKO of TIA1 and TIAL1 in HEK293 cells abrogates cell cycle progression and causes apoptotic cell death

A) Domain organization of human TIA1 family proteins. Protein length in amino acids (aa) is indicated. RRMs, RNA recognition motif; PrLD, prion-like domain. B) Verification of TIA1/TIAL1 DKO in HEK293 cells expressing either FH-tagged TIAL1 or TIA1 cultured with or without Dox. TUBB: Immunoblot (IB) for Tubulin beta chain. C) DKO/FH-TIAL1 and DKO/FH-TIA1 cells cultured without Dox (−Dox) over a 10-day time-course. D) Quantification of FH-tagged protein levels in DKO/FH-TIAL1 and DKO/FH-TIA1 cells upon Dox depletion based on IB (n=3) as shown in (B). E) Cell cycle analyses of DKO/FH-TIAL1 or DKO/FH-TIA1 cells cultured without Dox for up to 8 days. F) IB for phosphorylated H2B, cleaved CASP3 and cleaved PARP on DKO/FH-TIAL1 cell lysates cultured without Dox for up to 8 days. G) Fluorescence microscopy images of unsynchronized DKO/FH-TIAL1 cells cultured with (+Dox) or without Dox for 6 days (−Dox). Nuclei stained with DAPI. Scale bar, 2 μm. H) IF on TUBB to visualize centrosome formation in DKO/FH-TIAL1 cells. DAPI was used to stain nuclear DNA. Right panels depict colored overlays (DNA, blue; TUBB, green) of single grey scale images. Scale bar, 2 μm. I) For DKO/FH-TIAL1 cells cultured with (0 days) or without Dox (8 days), 100 mitotic cells were scored and the percentages of cells showing 1 (monopolar), 2 (bipolar asymmetric or symmetric), 3 (tripolar), or 4 (quadrupolar) centrosomes were calculated.

Figure 2. Loss of TIA1 function causes the activation of EIF2AK2-mediated stress response

A) RNA-FISH and IF on DKO/FH-TIAL1 cells cultured with (+Dox) or without Dox (−Dox 6 days) to visualize poly(A)-mRNAs, FH-tagged TIAL1 and G3BP1 (Meyer et al., 2016). Nuclei stained with DAPI. Scale bar, 20 μm. B) SG-positive DKO/FH-TIAL1 cells based on representative micrographs as shown in (A). C) Polysome profiles of DKO/FH-TIAL1 cells cultured without Dox for up to 8 days. D) IB for phosphorylated EIF2S1 or EIF2AK2/PKR on DKO/FH-TIAL1 cells cultured without Dox for up to 8 days. E) IB for phosphorylated EIF2S1 on TKO/FH-TIAL1 cells cultured without Dox for up to 8 days. Lysate of DKO/FH-TIAL1 cells cultured without Dox for 8 days served as control (loaded in the left lane). F) RNA-FISH and IF on TKO/FH-TIAL1 cells cultured with (+Dox) or without Dox (−Dox 6 days) to detect poly(A)-mRNAs and FH-tagged TIAL1. Scale bar, 20 μm. G) Cell cycle analyses of TKO/FH-TIAL1 or TKO/FH-TIA1 cells cultured without Dox for up to 8 days. H) Polysome profiles of TKO/FH-TIAL1 cells cultured without Dox for up to 8 days. I) Proliferation of DKO/FH-TIAL1 or TKO/FH-TIAL1 HEK293 cells cultured with or without Dox for up to 12 days.

Figure 3. TIA1 proteins bind to U-rich mRNAs in 3′ UTRs and intronic regions in proximity to 5′ and 3′ SSs

A) Autoradiograph and anti-HA IB of crosslinked and 5′-end radiolabeled RNA-FH-TIAL1 immunoprecipitates separated by SDS-PAGE following 4SU PAR-CLIP in HEK293 cells under normal or sodium arsenite stressed (60 min, 400 μM) growth conditions. XL, crosslinking; HC, heavy chain. B) Crosslinked TIAL1 PAR-CLIP reads with characteristic T-to-C conversions primarily map to the mRNA and precursor mRNA categories. C) PARalyzer-based distribution of binding sites resulting from FH-TIAL1 PAR-CLIP performed in HEK293 cells under normal growth conditions (Replicate N1). D–E) Normalized density of TIAL1 PAR-CLIP binding sites (red lines) over 3′ UTRs (D) or over introns (E) compared to a randomized background (grey lines). F) kmer-plot and MEME sequence logo representation of the TIAL1 RRE derived from PAR-CLIP binding sites in 3′ UTRs of target mRNAs. G) Overlap of target mRNAs crosslinked to TIAL1 or TIA1 proteins. H) Overlap of binding sites in the 3′ UTRs of target mRNAs crosslinked to TIAL1 or TIA1.

Figure 4. DKO of TIA1 proteins leads to upregulation of target mRNAs and aberrantly processed mRNAs

A) mRNA expression changes in DKO/FH-TIAL1 cells cultured as indicated and determined by poly(A)-RNA-seq. The empirical cumulative distribution function of TIAL1 PAR-CLIP targets binned by number of binding sites compared to non-targets (FPKM ≥3, black line). Dots on the x-axis indicate the median change. P values determined by the Mann-Whitney U-test. B) Types of AS detected from the poly(A)-RNA-seq data by MATS. Red boxes indicate alternatively spliced exons, blue boxes indicate constitutive exons. Numbers represent significant differential AS events (FDR<0.05) in DKO/FH-TIA1 or DKO/FH-TIAL1 cells cultured as indicated. Numbers in parentheses indicate number of significant events with higher inclusion level for sample 1 (+Dox) or for sample 2 (−Dox). SE, Skipped exon; MXE, Mutually exclusive exon; A5SS, Alternative 5′ SS; A3SS, Alternative 3′ SS. C) Overlap of mRNAs affected by AS in DKO/FH-TIA1 and DKO/FH-TIAL1 upon Dox depletion for 9 or 6 days, respectively. D) AS events in the SRSF3 or SRSF6 mRNAs reported by MATS shown with flanking exons. Histograms of poly(A)-mRNA-seq data represent exon read density; arcs represent splice junctions with number of reads mapped to the junction. Lower panel indicates TIAL1 PAR-CLIP sequence read coverage. E) RT-PCR results for the exon-inclusion and the exon-skipped forms occurring in SRSF3 and SRSF6 mRNAs upon depletion of Dox (up to 8 days) in DKO/FH-TIA1 or DKO/FH-TIAL1 cells, respectively. Blue and red boxes illustrate the alternative splice variants as shown in (D).

Figure 5. Loss of TIA1 family proteins causes processing defects of target mRNAs and reduction of protein levels

(A) Label-free quantification (LFQ) of cell lysates of DKO/FH-TIA1 HEK293 cells cultured as indicated. The heat maps represent unsupervised hierarchical clustering of LFQ intensities of proteins detected in all 5 samples with either more than 2-fold downregulation (left) or upregulation (right) upon Dox removal. (See also Table S5). B) IB for FH-TIA1, PRKRA, UFD1L, ALDH7A1, SRSF3, as well as SRSF6 of lysates of DKO/FH-TIAL1 cells. C) AS events detected in PRKRA, ALDH7A1, and UFD1L mRNAs upon depletion of Dox (up to 8 days) in DKO/FH-TIA1 or DKO/FH-TIAL1 cells by RT-PCR. D) Histograms representing the poly(A)-RNA-seq coverage of DKO/FH-TIA1 cells cultured for 0 (+Dox) or 8 (−Dox) days without Dox together with the depth of TIAL1 PAR-CLIP read coverage (d0 and d1 T-to-C) along the alternatively spliced exon 3 and the flanking exon 4 of the PRKRA mRNA.

Figure 6. TIA1 proteins are essential for accurate PRKRA mRNA processing to prevent EIF2AK2 activation

A) IB for phosphorylated EIF2AK2 and EIF2S1 on lysates of DKO/FH-TIAL1 cells expressing c-Myc-tagged PRKRA (PRKRA-MYC) or c-Myc alone (EMPTY-MYC) cultured with or without Dox. B) Proportion of SG-positive DKO/FH-TIAL1 and TKO/FH-TIAL1 cells as well as DKO/FH-TIAL1 cells either expressing PRKRA-MYC or EMPTY-MYC cultured as indicated. C) Domain organization of human PRKRA and truncated variants. DSRM, dsRNA-binding motif; Stau. C, Staufen C-terminal domain. D) IB for phosphorylated EIF2AK2 and EIF2S1 on lysates of DKO/FH-TIAL1 cells expressing PRKRA-MYC or c-Myc-tagged PRKRA mutants Δ1, Δ3, Δ12, Δ23 (as described in C) cultured with or without Dox. E) IB for phosphorylated EIF2AK2 on lysates of FH-tagged PRKRA expressing parental or PRKRA KO cells cultured with or without Dox. F) Localization of poly(A)-mRNAs as well as FH-tagged PRKRA and G3BP1 in FH-tagged PRKRA expressing PRKRA KO cells cultured with or without Dox by RNA-FISH and IF. Scale bar, 10 μm.

Figure 7

Simplified model depicting the function of TIA1 proteins described in this study.