Regular articleRho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis
Introduction
Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder characterized by the selective degeneration of motor neurons resulting in paralysis and death 3 to 5 years after onset in most patients (Strong et al., 2005). There are no treatments that will arrest disease progression and the cause remains elusive. Protein aggregate formation in motor neurons is a neuropathological hallmark of this disease. These aggregates contain proteins such as neurofilament (NF) (Kondo et al., 1986), peripherin (He and Hays, 2004), TAR DNA binding protein of 43 kDa (TDP-43) (Arai et al., 2006, Neumann et al., 2006), fused in sarcoma/translocated in liposarcoma (FUS/TLS) (Kwiatkowski et al., 2009, Vance et al., 2009), copper/zinc superoxide dismutase 1 (SOD1) (Shaw et al., 2008, Stieber et al., 2000), ubiquitin (Arai et al., 2006, Neumann et al., 2006), and 14-3-3 (Kawamoto et al., 2004). Additionally, motor neurons also show a selective decrease in the levels of polyadenylated mRNA, low molecular weight neurofilament (NFL) mRNA, α-internexin mRNA, and peripherin mRNA (Bergeron et al., 1994, Wong et al., 2000). These data, together with the participation of TDP-43 and FUS/TLS as RNA binding proteins (Buratti et al., 2001, Crozat et al., 1993), supports the hypothesis that alterations in RNA metabolism in motor neurons can lead to the development of protein aggregates in ALS (Strong, 2010). This hypothesis has been supported with a recent report showing a pathological association of an expanded hexanucleotide repeat in the noncoding region of C9ORF72 with ALS (Dejesus-Hernandez et al., 2011, Renton et al., 2011).
The NF proteins are a highly conserved family of neuronal intermediate filament proteins. There are 3 members of the NF family, a 68-kDa form (NFL), a 160-kDa form (middle molecular weight NF), and a 200-kDa form (high molecular weight NF). NF subunits assemble as heteropolymers in which the initial polymerization of NFL subunits is required for normal NF architecture formation (for review see Strong, 1999). In transgenic mouse models overexpressing peripherin or high molecular weight NF in which there is targeted disruption of the NFL gene, selective death of motor neurons is observed, along with motor dysfunction and the formation of intermediate filament aggregates, including intraneuronal NF aggregates (Beaulieu et al., 1999, Beaulieu et al., 2000). These results highlight the importance of NF stoichiometry in motor neurons and the importance of NFL mRNA stability as a possible cause of the protein aggregates observed in ALS (Szaro and Strong, 2010, Thyagarajan et al., 2007).
In mice, it has been observed that p190RhoGEF, a guanine nucleotide exchange factor (GEF) (van Horck et al., 2001) interacts with a destabilizing region of NFL mRNA providing stability to the transcript (Cañete-Soler et al., 2001). This protein is involved in NF protein aggregation observed in an RNA-triggered transgenic model of motor neuron disease (Lin et al., 2005, Nie et al., 2002). Previously, we showed that the RNA binding domain of the human homologue of p190RhoGEF, called rho guanine nucleotide exchange factor (RGNEF), can interact with NFL mRNA (Volkening et al., 2010). Here, we show that full length RGNEF is an RNA binding protein that acts as an NFL mRNA stability factor via 3′ untranslated region (UTR) destabilization and reduces NFL protein levels when overexpressed in HEK293T cells. Moreover, we show that RGNEF also presents a pathogenic phenotype including aggregate formation in motor neurons of ALS patients.
Section snippets
Cases
Postmortem frozen and formalin-fixed, paraffin-embedded tissues were collected as part of the ALS protocol at London Health Sciences Centre (London, Ontario, Canada). Six control cases (4 male, 2 female, age range 61–74 years), 13 sporadic ALS (sALS; 8 male, 5 female, age range 45–80 years) cases, and 3 familial ALS (fALS; 2 male, 1 female, age range 56–71 years) cases without known mutations in the coding sequence of SOD1, FUS/TLS, and TDP-43 and without C9ORF72 expanded repeats were used in
Human RGNEF shows a high similarity with murine p190RhoGEF
An amino acid (aa) sequence comparison analysis was performed to determine the similarity between the murine p190RhoGEF (GenBank U73199) and its human homolog, RGNEF (GenBank BC157846). These proteins share the same conserved domains (Fig. 1A). After Basic Local Alignment Search Tool analysis (BLAST utility; blast.ncbi.nlm.nih.gov), we observed an 80% identity between p190RhoGEF and RGNEF at the protein level. The GEF domain (mouse: aa 847–1039; human: aa 850–1042 according the conserved
Discussion
The major finding in this study is that RGNEF, the human homolog of the mouse protein p190RhoGEF, is a bifunctional protein that appears to be involved in the pathology of ALS. This manifests as aggregates and skeins that colocalize with other proteins that have been described as pathological in ALS (TDP-43, ubiquitin, p62/Sequestosome-1). We have shown that RGNEF is an RNA binding protein that acts as an RNA destabilizing factor for human NFL mRNA via its 3′ UTR, and can regulate NFL protein
Disclosure statement
The authors declare that they have no competing interests.
Acknowledgements
The authors thank Dr. Rosa Rademakers for the C9ORF72 expanded repeats genotyping of ALS patients and Dr. Wouter H. Moolenaar for kindly providing pcDNA-p190RhoGEF plasmid. This work was supported by the Canadian Institute of Health Research (CIHR), The McFeat Family Fund, and the ALS Society of Canada.
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2020, Neurobiology of AgingCitation Excerpt :An early study showed that p190RhoGEF modulates murine NFL mRNA stability and is involved in aggregation of NFL protein, which is an upstream event triggering neurotoxicity in MN disease (Lin et al., 2005). Moreover, a direct interaction between NFL mRNA and RGNEF, as well as the pathogenic cytoplasmic inclusions of RGNEF were observed in MNs of patients with ALS, coexisted with or without C9orf72 expansion (Droppelmann et al., 2013; Volkening et al., 2010), suggesting that RGNEF pathology can, independently or synergistically with other RNA-binding proteins, contribute to ALS. We identified 2 LoF mutations (p.Ser531Ter and p.Lys1070fs) in ALS cases, both locate upstream of the protein-protein binding (including FAK-, RNA-, and microtubule-binding) domains and can cause complete depletion or disruption of its binding activity.
A novel overlapping NLS/NES region within the PH domain of Rho Guanine Nucleotide Exchange Factor (RGNEF) regulates its nuclear-cytoplasmic localization
2019, European Journal of Cell BiologyCitation Excerpt :To analyze the functionality of this putative NLS signal, we created two mutant constructs of RGNEF: pcDNA-RGNEF-ΔPH-myc which included a deletion of the 119 residues of the PH domain (del 1084–1202) and pcDNA-RGNEF-mutNLS-myc in which four basic residues of the putative NLS were point-mutated to neutral alanine residues (R1101 A, K1103 A, K1120 A, and K1123 A) without inducing major changes in the predicted folding of the protein (Fig. 2A and Fig S1A). HEK293 T cells transfected with the vector expressing wild-type (wt) RGNEF (pcDNA-RGNEF-myc) showed predominantly cytoplasmic localization of the protein with moderate levels of nuclear localization (Fig. 2B), consistent with what we observed previously (Droppelmann et al., 2013). Both NLS lacking constructs showed protein largely absent from the nucleus (Fig. 2B).
Rho guanine nucleotide exchange factor (RGNEF) is a prosurvival factor under stress conditions
2017, Molecular and Cellular NeuroscienceCitation Excerpt :All the work with animals was performed following the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and as approved by the Animal Use Subcommittee of the Western University Council on Animal Care. The construct pcDNA-RGNEF-myc expressing a myc-tagged full length RGNEF protein was previously generated by our group (Droppelmann et al. 2013a). In addition, we generated six RGNEF constructs with deletions in several domains by standard molecular biology procedures (Supplementary Fig. 1 and Supplementary Table 1).