Analysis of Nuclear Export Sequence Regions of FUS-Related RNA-Binding Proteins in Essential Tremor

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the degeneration of both upper and lower motor neurons in the brain and spinal cord. ALS is associated with protein misfolding and inclusion formation involving RNA-binding proteins, including TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS). The 125-kDa Matrin3 is a highly conserved nuclear DNA/RNA-binding protein that is implicated in many cellular processes, including binding and stabilizing mRNA, regulating mRNA nuclear export, modulating alternative splicing, and managing chromosomal distribution. Mutations in MATR3, the gene encoding Matrin3, have been identified as causal in familial ALS (fALS). Matrin3 lacks a prion-like domain that characterizes many other ALS-associated RNA-binding proteins, including TDP-43 and FUS, however, our bioinformatics analyses and preliminary studies document that Matrin3 contains long intrinsically disordered regions that may facilitate promiscuous interactions with many proteins and may contribute to its misfolding. In addition, these disordered regions in Matrin3 undergo numerous post-translational modifications, including phosphorylation, ubiquitination and acetylation that modulate the function and misfolding of the protein. Here we discuss the disordered nature of Matrin3 and review the factors that may promote its misfolding and aggregation, two elements that might explain its role in ALS pathogenesis.

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Trypanosoma brucei RNA Binding Proteins p34 and p37 Mediate NOPP44/46 Cellular Localization via the Exportin 1 Nuclear Export Pathway

Eukaryotic Cell ◽

10.1128/ec.00176-07 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2206-2213 ◽

Cited By ~ 12

Author(s):

Kristina Hellman ◽

Kimberly Prohaska ◽

Noreen Williams

Keyword(s):

Amino Acid ◽

Trypanosoma Brucei ◽

Nuclear Export ◽

Cellular Localization ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Export Signal ◽

Amino Acid Sequences ◽

Protein Levels

ABSTRACT We have previously identified and characterized two novel nuclear RNA binding proteins, p34 and p37, which have been shown to interact with a family of nucleolar phosphoproteins, NOPP44/46, in Trypanosoma brucei. These proteins are nearly identical, the major difference being an 18-amino-acid insert in the N terminus of p37. In order to characterize the interaction between p34 and p37 and NOPP44/46, we have utilized an RNA interference (RNAi) cell line that specifically targets p34 and p37. Within these RNAi cells, we detected a disruption of a higher-molecular-weight complex containing NOPP44/46, as well as a dramatic increase in nuclear NOPP44/46 protein levels. We demonstrated that no change occurred in NOPP44/46 mRNA steady-state levels or stability, nor was there a change in cellular protein levels. These results led us to investigate whether p34 and p37 regulate NOPP44/46 cellular localization. Examination of the p34 and p37 amino acid sequences revealed a leucine-rich nuclear export signal, which interacts with the nuclear export factor exportin 1. Immune capture experiments demonstrated that p34, p37, and NOPP44/46 associate with exportin 1. When these experiments were performed with p34/p37 RNAi cells, NOPP44/46 no longer associated with exportin 1. Sequential immune capture experiments demonstrated that p34, p37, NOPP44/46, and exportin 1 exist in a common complex. Inhibiting exportin 1-mediated nuclear export led to an increase in nuclear NOPP44/46 proteins, indicating that they are exported from the nucleus via this pathway. Together, our results demonstrate that p34 and p37 regulate NOPP44/46 cellular localization by facilitating their association with exportin 1.

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Abstract P-22: Enhanced Crosslinking and Immunoprecipitation (Eclip) Data Reveal Interactions of RNA Binding Proteins with the Human Ribosome

International Journal of Biomedicine ◽

10.21103/ijbm.11.suppl_1.p22 ◽

2021 ◽

Vol 11 (Suppl_1) ◽

pp. S21-S21

Author(s):

Andrey Buyan ◽

Ivan Kulakovskiy ◽

Sergey Dmitriev

Keyword(s):

Translational Control ◽

Nuclear Export ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Repetitive Sequences ◽

Structural Data

Background: The ribosome is a protein-synthesizing molecular machine composed of four ribosomal RNAs (rRNAs) and dozens of ribosomal proteins. In mammals, the ribosome has a complicated structure with an additional outer layer of rRNA, including large tentacle-like extensions. A number of RNA binding proteins (RBPs) interact with this layer to assist ribosome biogenesis, nuclear export and decay, or to modulate translation. Plenty of methods have been developed in the last decade in order to study such protein-RNA interactions, including RNA pulldown and crosslinking-immunoprecipitation (CLIP) assays. Methods: In the current study, using publicly available data of the enhanced CLIP (eCLIP) experiments for 223 proteins studied in the ENCODE project, we found a number of RBPs that bind rRNAs in human cells. To locate their binding sites in rRNAs, we used a newly developed computational protocol for mapping and evaluation of the eCLIP data with the respect to the repetitive sequences. Results: For two proteins with known ribosomal localization, uS3/RPS3 and uS17/RPS11, the identified sites were in good agreement with structural data, thus validating our approach. Then, we identified rRNA contacts of overall 22 RBPs involved in rRNA processing and ribosome maturation (DDX21, DDX51, DDX52, NIP7, SBDS, UTP18, UTP3, WDR3, and WDR43), translational control during stress (SERBP1, G3BP1, SND1), IRES activity (PCBP1/hnRNPE1), and other translation-related functions. In many cases, the identified proteins interact with the rRNA expansion segments (ES) of the human ribosome pointing to their important role in protein synthesis. Conclusion: Our study identifies a number of RBPs as interacting partners of the human ribosome and sheds light on the role of rRNA expansion segments in translation.

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Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins

The Journal of Cell Biology ◽

10.1083/jcb.200110082 ◽

2002 ◽

Vol 156 (1) ◽

pp. 53-64 ◽

Cited By ~ 108

Author(s):

Amy M. Brownawell ◽

Ian G. Macara

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dependent Manner ◽

Intact Cells ◽

Double Stranded Rna ◽

Permeabilized Cells ◽

Dsrna Binding ◽

Rna Export

We have identified a novel human karyopherin (Kap)β family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.

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Emerging Roles of RNA-Binding Proteins in Seed Development and Performance

International Journal of Molecular Sciences ◽

10.3390/ijms21186822 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6822

Author(s):

Lijuan Lou ◽

Ling Ding ◽

Tao Wang ◽

Yong Xiang

Keyword(s):

Seed Development ◽

Translational Control ◽

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Research Progress ◽

Mrna Metabolism ◽

And Performance ◽

Post Transcriptional Regulation

Seed development, dormancy, and germination are key physiological events that are not only important for seed generation, survival, and dispersal, but also contribute to agricultural production. RNA-binding proteins (RBPs) directly interact with target mRNAs and fine-tune mRNA metabolism by governing post-transcriptional regulation, including RNA processing, intron splicing, nuclear export, trafficking, stability/decay, and translational control. Recent studies have functionally characterized increasing numbers of diverse RBPs and shown that they participate in seed development and performance, providing significant insight into the role of RBP–mRNA interactions in seed processes. In this review, we discuss recent research progress on newly defined RBPs that have crucial roles in RNA metabolism and affect seed development, dormancy, and germination.

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Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis

Brain ◽

10.1093/brain/awz217 ◽

2019 ◽

Vol 142 (9) ◽

pp. 2572-2580 ◽

Cited By ~ 34

Author(s):

Giulia E Tyzack ◽

Raphaelle Luisier ◽

Doaa M Taha ◽

Jacob Neeves ◽

Miha Modic ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Spinal Cord Tissue ◽

Cytoplasmic Inclusions ◽

Sporadic Als ◽

Lateral Sclerosis ◽

Fus Protein

Abstract Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

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Nucleocytoplasmic Shuttling of JAZ, a New Cargo Protein for Exportin-5

Molecular and Cellular Biology ◽

10.1128/mcb.24.15.6608-6619.2004 ◽

2004 ◽

Vol 24 (15) ◽

pp. 6608-6619 ◽

Cited By ~ 39

Author(s):

Ting Chen ◽

Amy M. Brownawell ◽

Ian G. Macara

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Accumulation ◽

Facilitated Diffusion ◽

Dependent Manner ◽

Permeabilized Cells ◽

Dsrna Binding ◽

Ran Gtp

ABSTRACT Exportin-5 is a nuclear export receptor for certain classes of double-stranded RNA (dsRNA), including pre-micro-RNAs, viral hairpin RNAs, and some tRNAs. It can also export the RNA binding proteins ILF3 and elongation factor EF1A. However, the rules that determine which RNA binding proteins are exportin-5 cargoes remain unclear. JAZ possesses an unusual dsRNA binding domain consisting of multiple C2H2 zinc fingers. We found that JAZ binds to exportin-5 in a Ran-GTP- and dsRNA-dependent manner. Exportin-5 stimulates JAZ shuttling, and gene silencing of exportin-5 reduces shuttling. Recombinant exportin-5 also stimulates nuclear export of JAZ in permeabilized cells. JAZ also binds to ILF3, and surprisingly, this interaction is RNA independent, even though it requires the dsRNA binding domains of ILF3. Exportin-5, JAZ, and ILF3 can form a heteromeric complex with Ran-GTP and dsRNA, and JAZ increases ILF3 binding to exportin-5. JAZ does not contain a classical nuclear localization signal, and in digitonin-permeabilized cells, nuclear accumulation of JAZ does not require energy or cytosol. Nonetheless, low temperatures prevent JAZ import, suggesting that nuclear entry does not occur via simple diffusion. Together, these data suggest that JAZ is exported by exportin-5 but translocates back into nuclei by a facilitated diffusion mechanism.

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HIV-1 RNA genomes initiate host protein packaging in the cytosol independently of Gag capsid proteins

10.1101/846105 ◽

2019 ◽

Author(s):

Jordan T. Becker ◽

Edward L. Evans ◽

Bayleigh E. Benner ◽

Stephanie L. Pritzl ◽

Laura E. Smith ◽

...

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Host Protein ◽

Capsid Proteins ◽

Particle Assembly ◽

Nonsense Mediated Decay ◽

Genome Interactions ◽

Hiv 1

ABSTRACTHIV-1 RNA genomes interact with diverse RNA binding proteins (RBPs) in the cytoplasm including antiviral factor APOBEC3G (A3G) that, in the absence of viral Vif proteins, is packaged into virions. Where and when HIV-1-A3G interactions are initiated for packaging inside the cell is unknown, and the relative contributions of genome vs. Gag capsid proteins to this process remains controversial. Here we visualized A3G responses to HIV-1 infection over an entire replication cycle using long-term (up to 72 h) live single cell imaging. We show that Vif-deficient HIV-1 dramatically shifts A3G and a second RNA surveillance factor, MOV10, from the cytoplasm to virus particle assembly sites with little to no net discernible effects on general markers of cytoplasmic processing bodies (DCP1A), stress granules (TIA-1), or a marker of the nonsense-mediated decay machinery (UPF1). Using a new live cell RNA-protein interaction assay based on RNA tethering (the in-cell RNA-protein interaction protocol, or IC-IP), we provide evidence that A3G- and MOV10- genome interactions are selective, strong, occur in presence or absence of Gag, and are initiated in the cytosol soon if not immediately after genome nuclear export. Finally, although Gag is sufficient to package A3G into virions even in the absence of genomes, single virion imaging indicates that selective A3G-genome interactions promote much more consistent per virion delivery of A3G to assembly sites. Collectively, these studies suggest a paradigm for early, strong, and persistent cytosolic detection of select HIV-1 RNA signatures by A3G, MOV10 and other host RBPs that are enriched in virions.IMPORTANCEHost-pathogen interactions determine the success of viral replication. While extensive work has identified many interactions between HIV-1 and cellular factors, our understanding of where these interactions in cells occur during the course of infection is incomplete. Here, we show that multiple RNA-binding proteins (including the antiviral restriction factor, APOBEC3G, and MOV10 helicase) bind HIV-1 RNA genomes in the cytoplasm and co-traffic with them into progeny virions. Furthermore, we show that these interactions with HIV-1 RNA occur in the absence of Gag and are sufficiently strong to recruit these to otherwise non-native subcellular locales. Together, these data begin to illuminate the intracellular trafficking pathways shared by host RNA binding proteins and the viral RNAs they preferentially bind.

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Flanking sequences regulate the toxicity, non-ATG translation, and aggregation of RNA with tandem CAG Repeats

10.1101/2021.08.30.458106 ◽

2021 ◽

Author(s):

Michael R. Das ◽

Yeonji Chang ◽

Reuben Saunders ◽

Nan Zhang ◽

Colson Tomberlin ◽

...

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cag Repeats ◽

Cell Toxicity ◽

Cis Acting ◽

Cytoplasmic Rna ◽

Flanking Sequences ◽

Ran Translation

Nucleotide repeat-expansions cause several neurodegenerative disorders, including Huntington's disease and spinocerebellar ataxia. The expanded repeat-containing RNA transcribed from the affected loci agglomerate in the nucleus as pathogenic foci. Here we demonstrate that depending on their surrounding sequence context, RNAs with expanded CAG repeats can also undergo nuclear export and aggregate in the cytoplasm. Cytoplasmic aggregation of repeat-containing RNA coincides with several disease hallmarks, including repeat-associated non-AUG (RAN) translation, mislocalization of RNA binding proteins, and cell toxicity. Interestingly, the repeat-containing RNA co-aggregate with RAN translation products. Inhibition of RAN translation prevents cytoplasmic RNA aggregation and also alleviates cell toxicity. Our findings provide a cogent explanation for aberrant cytoplasmic localization of RNA binding proteins and implicate cis-acting flanking sequences in mediating RAN translation and disease.

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