[PubMed] [CrossRef] [Google Scholar] 54. OPA1 or overexpression of OPA1v5 provoked mitochondrial clustering. Middle-term expression of TIA1b/TIARb triggers reactive oxygen species production and mitochondrial DNA damage, which is accompanied by mitophagy, autophagy, and apoptosis. In contrast, HuR expression promotes mitochondrion-dependent cell proliferation. Collectively, these results provide molecular insights into the antagonistic functions of TIA1b/TIARb and HuR in mitochondrial activity dynamics and suggest that their balance might contribute to mitochondrial physiopathology. mitochondrial biogenesis, the dynamic equilibrium between fusion and fission events and associated inner membrane remodeling are fundamental for mitochondrial morphology and function in eukaryotic cells (2). The inner mitochondrial membrane is extensively folded, producing invaginations called cristae, which control oxidative phosphorylation (OXPHOS), electron and metabolite transport, and cell death and survival (3). Consequently, mitochondrial network dysregulation is linked to several pathophysiological conditions, including cancer, diabetes, and neurodegenerative diseases (4,C8). Mitochondrial biogenesis is controlled by specific transcription factors, such as peroxisome proliferator-activated receptor coactivator 1 (PGC-1) (9), which activates mitochondrial DNA (mtDNA) replication, and by RNA binding proteins (RBPs), such as, for example, Pioglitazone hydrochloride Y-box-binding protein 1 (YB-1) and clustered mitochondria (cluA/CLU1) homolog (CLUH), which exert posttranscriptional control (10, 11). Conversely, mitochondrial dynamics is controlled by mitochondrion-shaping proteins that regulate fusion Pioglitazone hydrochloride and fission events. Core components of the mitochondrial fusion/fission machinery include mitofusin 1 (MFN1), mitofusin 2 (MFN2), and optic atrophy 1 (OPA1), which promote fusion, whereas Pioglitazone hydrochloride fission is governed by dynamin-related protein 1 (DRP1) and by adaptor proteins such as mitochondrial fission factor (MFF), mitochondrial dynamics proteins (MiD49 and MiD51), and fission 1 (FIS1) (1,C8). T-cell intracellular antigen 1 (TIA1), TIA1-like/related protein (TIAL1/TIAR), and Hu antigen R (HuR/ELAVL1) are RBPs that exert transcriptional and/or posttranscriptional control of gene expression (12,C23). Their regulatory roles are directed at specific sites within the transcriptome through association with specific RNA sequence motifs (13,C20). TIA1 and TIAR have two main isoforms generated by alternative splicing of their pre-mRNAs. The EFNB2 TIA1a isoform (43 kDa) differs from isoform TIA1b (40 kDa) by inclusion of an 11-amino-acid sequence, encoded by exon 5 (12). Isoform TIARa (50 kDa) differs from isoform TIARb (42 kDa) in that it contains a sequence of 17 amino acids, encoded by the last 51 nucleotides of exon 3 (12). In the nucleus, TIA and HuR proteins modulate DNA-dependent transcription and processing of the precursor RNAs (i.e., constitutive and alternative splicing), and in the cytoplasm, they regulate localization, stability, and/or translation of mRNAs. Their roles are critical for cell homeostasis, since they control the expression of essential genes in biological programs such as Pioglitazone hydrochloride survival and death, proliferation and differentiation, inflammation, environmental stress, viral infections, embryogenesis, tumorigenesis, and aging, with an impact in physiopathology (12,C18, 20, 21). Further, they seem to have an important role during embryogenesis, since mice deficient for TIA1, TIAR, or HuR (as well as ectopic overexpression of TIAR) have high rates of embryonic and postnatal lethality (24,C27). Here we show that ectopic expression of TIA1b, TIARb, and HuR alters mitochondrial morphology and function by targeting, differentially and antagonistically, regulatory events associated with transcriptional and/or posttranscriptional control of OPA1 gene expression. RESULTS TIA1b, TIARb, and HuR proteins have opposing effects in mitochondrial dynamics. Inducible HEK293 cells expressing green fluorescent protein (GFP), GFP-TIA1b, GFP-TIARb, or GFP-HuR (FT293 cells) were generated using the Flp-In T-REx platform (28) and analyzed by Western blotting (Fig. 1A). Ectopic expression of TIA1b or TIARb isoforms for 24 to 48 h resulted in a disorganization of the mitochondrial network as assessed by confocal microscopy using an antibody against Tom20 (Fig. 1A) and by Mitotracker staining (data not shown). Compared with control FT293 cells, expression of either TIA1b or TIARb led to changes in mitochondrial spatial dynamics without observable changes in mitochondrial mass (Fig. 1A and ?andB).B). In contrast, cells expressing HuR presented enlarged mitochondria that were distributed throughout the cell cytoplasm (Fig. 1A and ?andB).B). Detailed analysis by transmission electron microscopy (TEM) revealed that whereas TIA1b and TIARb expression promoted mitochondrial clustering and fission, HuR expression provoked mitochondrial fusion (Fig. 1B). Closer inspection of mitochondria revealed evident changes in crista organization in TIA1b- and TIARb-expressing cells, with many cristae having a slightly wider and more loosely organized intermembrane space.