The expression of Welander distal myopathy-related TIA1 mutation exacerbates the dynamics of P-bodies and stress granules under oxidative stress

T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein that is primarily involved in the post-transcriptional control of the cellular fate of RNAs. Moreover, it is a key component of stress granules (SGs), RNA and protein aggregates that are formed in response to stressful stimuli to reduce cellular activity as a survival mechanism. TIA1 p.E384K mutation is the genetic cause of Welander distal myopathy (WDM), a late-onset muscular dystrophy whose pathogenesis has been related to modify SGs dynamics. In this study, we have focused on the differential transcriptomic and proteomic characterization of purified wild-type (WT) and WDM TIA1-dependent SGs by affinity immunopurification followed by mass spectrometry and RNA seq analysis under oxidative stress. The results have revealed small differences in specific components of cellular machineries involving in regulating splicing, RNA degradation and/or translation linked to WDM TIA1 expression under oxidative stress. Our findings suggest a potential regulatory linking among WDM TIA1-dependent SGs and the number, size and colocalization/overlapping with P-bodies under oxidative stress. Taken together, these findings allow us to expand our knowledge on the role of Welander's TIA1 mutation in interfacing SGs and P-bodies dynamics by involving potential regulatory impacts on cell machineries of RNA decay/turnover and/or translation. Overall design: Stress granules cores were enriched from FT293-TIA1aWT and FT293-TIA1aWDM cells stably transfected with plasmids for GFP-TIA1aWT and GFP-TIA1aWDM expression (generated in-house) as described previously (Sánchez-Jiménez et al. (2015) Cell Death Dis., 6, e1669; Carrascoso et al. (2019) Mol. Cell. Biol., 39, e0029918). Briefly, FT293 cells expressing either GFP-TIA1aWT or GFP-TIA1aWDM were subjected to 0.5 mM NaAsO2 for 1 h and snap-frozen on dishes. Cells were scraped, washed once with DMEN alone, snap-frozen with liquid nitrogen and stored at -70ºC. Cellular pellets were thawed on ice at 4ºC for 10 min and processed using lysis buffer (50 mM Tris HCl, pH 7.4, 100 mM KOAc, 2 mM MgOAc, 0.5 mM DTT, 50 µg/ml heparin, 0.5% NP-40, and protease inhibitors cocktail) and passed through a 25G x 5/8” (0.5mm x 16 mm) (BD Microlance 3) needle. The lysate was centrifuged at 1,000 g for 5 min, and the supernatant from this spin was centrifuged at 16,873 g for 20 min. The pellet containing SG cores was resuspended in the lysis buffer and incubated with GFP-Trap magnetic agarose beads (Chromotek) for 4 h. Beads were washed three times with washing buffer 1 (20 mM Tris HCl and 200 mM NaCl, pH 8.0), once with washing buffer 2 (20 mM Tris HCl and 500 mM NaCl, pH 8.0), and once with washing buffer 3 (lysis buffer containing 2 M urea). The proteins were eluted in 2x Laemmli sample buffer at 95ºC for 10 min. The total RNAs were purified using TRIzolR Reagent (Ambion by Life technologies).