Developmentally Regulated RNA-binding Protein 1 (Drb1)/RNA-binding Motif Protein 45 (RBM45), a Nuclear-Cytoplasmic Trafficking Protein, Forms TAR DNA-binding Protein 43 (TDP-43)-mediated Cytoplasmic Aggregates

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 nuclearcytoplasmic trafficking induces toxic cytoplasmic aggregates, suggesting that mislocalization of Drb1 is involved in the cause of cytotoxicity in neuronal cells. 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. Although 5-10% of ALS cases are familial, ϳ90% of cases are sporadic with unknown etiology (1). In contrast, frontotemporal lobar degeneration (FTLD) is a group of neurodegenerative diseases that affect frontal and temporal cortices with characteristic cognitive defects such as personality and behavioral changes as well as progressive deterioration of language skills (2). A subgroup of patients presents overlapped clinical characteristics of ALS and FTLD. Cytoplasmic inclusion bodies in neuronal cells are a common pathological hallmark in ALS and FTLD, implying that a common etiological pathway exists in ALS and FTLD pathogenesis (1, 3). TAR DNA-binding protein 43 (TDP-43), an RNA-binding protein, was the first protein identified in cytoplasmic inclusion bodies in most sporadic ALS and several FTLD cases (4, 5). Since then, other RNA-binding proteins, such as fused in sarcoma/translated in liposarcoma (FUS/TLS), and heterogeneous nuclear ribonucleoproteins were also identified as components of cytoplasmic inclusion bodies. Because disease-associated mutations in the TDP-43 or FUS/TLS gene were identified in several familial ALS and F...

RBM45 associates with nuclear stress bodies and forms nuclear inclusions during chronic cellular stress and in neurodegenerative diseases

“…This suggests that ongoing cellular stressors or pathological processes alter the normal diffuse nuclear localization of RBM45. RBM45 interacts with both TDP-43 and FUS 3 , is found in TDP-43-positive neuronal cytoplasmic inclusions in ALS, FTLD, and AD, and has previously been reported to associate with cytoplasmic SGs 3,4 . We examined the distribution of RBM45 during conditions of cellular stress and found no evidence of RBM45 cytoplasmic translocation or association with SGs ( Figure 1D-E and Figure 2).…”

“…To better understand the normal functions RBM45 and how these relate to inclusion formation, we previously characterized functional protein domains in the RBM45 protein. By virtue of a bipartite nuclear localization sequence (NLS), RBM45 is a predominantly nuclear protein under basal conditions 3,4,5 . Mutation of the RBM45 NLS sequence leads to cytoplasmic sequestration of the protein, with attendant incorporation into cytoplasmic stress granules (SGs) 3,4 .…”

“…By virtue of a bipartite nuclear localization sequence (NLS), RBM45 is a predominantly nuclear protein under basal conditions 3,4,5 . Mutation of the RBM45 NLS sequence leads to cytoplasmic sequestration of the protein, with attendant incorporation into cytoplasmic stress granules (SGs) 3,4 . RBM45 also contains 3 RNA recognition motifs (RRMs) that govern its binding to RNA sequences 3,4,6 .…”

“…Mutation of the RBM45 NLS sequence leads to cytoplasmic sequestration of the protein, with attendant incorporation into cytoplasmic stress granules (SGs) 3,4 . RBM45 also contains 3 RNA recognition motifs (RRMs) that govern its binding to RNA sequences 3,4,6 .…”

Molecular Genetic Analysis of Rbm45/Drbp1: Genomic Structure, Expression, and Evolution

“…RNA-binding motif protein 45 (Rbm45), also known as developmentally regulated RNAbinding protein 1(Drbp1), was first identified as a novel RNA-binding protein through the screening of a rat cerebral expression library with polyclonal antibody against consensus RRM sequences. Rbm45 has three (Li et al, 2015b;Mashiko et al, 2016) or four canonical RBDs (Tamada et al, 2002;HomoloGene, https://www.ncbi.nlm.nih.gov/homologene/?term=Rbm45 [accessed 11.15.2017]) and is expressed in a spatiotemporal manner in developing rat brain, with highest expression, as measured by mRNA accumulation, occurring between embryonic days 12 to 16. There is a subsequent decline in expression of Rbm45 during late embryonic development that extends into adulthood as neuronal precursor cells differentiate (Tamada et al, 2002).…”