Abstract
The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts.
Highlights
The trans-activating response region DNA-binding protein 43 (TDP-43) is a member of the family of heterogeneous nuclear ribonucleoproteins and contains two highly conserved RNA recognition motifs (RRMs) and a non-conserved C-terminal region that mediates protein-protein interactions[1]
We established human Flp-InTM T-RExTM 293 (T-REx 293) cell line expressing doxycycline-inducible wild-type human TDP-43 carrying a triple affinity-purification tag (DAP-tag: 6 × histidine, biotin, and FLAG; Supplementary Fig. 1a), and showed that the DAP-TDP-43 expression reduced the level of endogenous TDP-43, suggesting that the exogenous DAP-TDP-43 is behaved as native TDP-43 by constitutive negative feedback mechanism (Supplementary Fig. 1b)[4]
We confirmed the association of endogenous TDP-43 with these mt-tRNAs using anti-TDP-43 immunoprecipitation (Fig. 1c), and the interactions were determined to occur directly within intact cells as assessed with UV cross-linking in vivo and immunoprecipitation (Fig. 1d)
Summary
The trans-activating response region DNA-binding protein 43 (TDP-43) is a member of the family of heterogeneous nuclear ribonucleoproteins and contains two highly conserved RNA recognition motifs (RRMs) and a non-conserved C-terminal region that mediates protein-protein interactions[1]. The mitochondrial fusion protein mitofusin 2 (Mfn2) has been demonstrated to be involved in mitochondrial impairment, but the functional relationship between TDP-43 and Mfn[2] is unknown[27]. In this regard, TDP-43 mutants disrupt the vesicle-associated membrane protein-associated protein B (VAPB)– protein tyrosine phosphatase-interacting protein-51 (PTPIP51) interaction and cellular Ca2+ homeostasis, which disrupts mitochondria–endoplasmic reticulum interactions that are implicated in several physiological processes including ATP production, mitochondrial biogenesis, and apoptosis[29]; TDP43 is involved only indirectly in these processes through its ability to activate glycogen synthase kinase-3β29. Despite the obvious involvement of TDP-43 in mitochondrial function, the direct mitochondrial target of TDP-43 remains elusive
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