Abstract
Cytoplasmic protein aggregates are one of the pathological hallmarks of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Several RNA-binding proteins have been identified as components of inclusion bodies. Developmentally regulated RNA-binding protein 1 (Drb1)/RNA-binding motif protein 45 is an RNA-binding protein that was recently described as a component in ALS- and FTLD-related inclusion bodies. However, the molecular mechanism underlying cytoplasmic Drb1 aggregation remains unclear. Here, using an in vitro cellular model, we demonstrated that Drb1 co-localizes with cytoplasmic aggregates mediated by TAR DNA-binding protein 43, a major component of ALS and FTLD-related inclusion bodies. We also defined the domains involved in the subcellular localization of Drb1 to clarify the role of Drb1 in the formation of cytoplasmic aggregates in ALS and FTLD. Drb1 predominantly localized in the nucleus via a classical nuclear localization signal in its carboxyl terminus and is a shuttling protein between the nucleus and cytoplasm. Furthermore, we identify a double leucine motif serving as a nuclear export signal. The Drb1 mutant, presenting mutations in both nuclear localization signal and nuclear export signal, is prone to aggregate in the cytoplasm. The mutant Drb1-induced cytoplasmic aggregates not only recruit TAR DNA-binding protein 43 but also decrease the mitochondrial membrane potential. Taken together, these results indicate that perturbation of Drb1 nuclear-cytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells.
Highlights
amyotrophic lateral sclerosis (ALS),2 one of the most devastating and untreatable neurodegenerative diseases, affects motor neurons selectively with relentless progression and causes respiratory failure within 2–5 years from onset
developmentally regulated RNAbinding protein 1 (Drb1) Co-localizes with TAR DNA-binding protein 43 (TDP-43) Mutant-induced Cytoplasmic Aggregates—To elucidate the mechanism underlying the co-localization of Drb1 into TDP-43-positive cytoplasmic inclusions in ALS pathology, we established a cellular model of HeLa cells to form disease-like cytoplasmic aggregates using EGFP-fused TDP-43 (Fig. 1A)
When cells were co-transfected with T7-Drb1-WT and EGFP-TDP-mt, T7-Drb1-WT co-localized with TDP-mt-induced cytoplasmic aggregates and presented a partial nucleoplasmic co-localization (Fig. 1B, j–l)
Summary
Plasmid Construction—The plasmids used in this study were produced in the pcDNA3 (Invitrogen), pEF-Bos-T7 [25], pEGFP (Clontech, Tokyo, Japan), and pGEX-6p-1 (GE Healthcare) vectors, and each was fused with EGFP, Clover (Addgene plasmid 40259), a T7 tag, a myc tag, or GST at the N terminus and with mRuby (Addgene plasmid 40260) at the C terminus. A deletion mutant of Clover-T7-tag-Drb 400 was generated by PCR using pcDNA3-Clover-T7-tag-Drb1-WT as a template with a pair of primers (T7-XbaF (forward) and 5Ј-CGGTCTAGACTAAGGATGAGGATTAAACACAATA-3Ј (reverse); the underline represents the XbaI site) to insert the mutated cDNA into the XbaI site of pcDNA3-Clover. The deletion mutants of pEF-Bos-T7-Drb were generated by PCR using pcDNA3-Clover-T7-tag-Drb1-WT as a template with the following primer pairs to insert the mutated cDNA into the BamHI site of pEF-Bos-T7: 401– 474, 5Ј-CGAGGATCCCTTTACCTTTAGACGTATTAGA-3Ј (forward) and Drb-BamR (reverse); 2–300, Drb-BamF (forward) and 5ЈATGGATCCTCATGCATAAATAGCTGATGC-3Ј (reverse); 301– 474, 5Ј-GCAGGATCCAAAAATACAAATTACATGGATT-3Ј (forward) and Drb-BamR (reverse); and 301– 400, 5ЈGCAGGATCCAAAAATACAAATTACATGGATT-3Ј (forward) and 5Ј-CGGGGATCCTAAGGATGAGGATTAAACACAATA-3Ј (reverse); the underlines represent BamHI sites. HeLa cells overexpressing Clover or Clover-Drb fusion protein (WT, R470G, or mtLL/ R470G) were plated in poly-L-lysine-coated 35-mm glass bottom dishes, and the following assay was performed.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
