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

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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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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Modulation of MicroRNA Processing by Dicer via Its Associated dsRNA Binding Proteins

Non-Coding RNA ◽

10.3390/ncrna7030057 ◽

2021 ◽

Vol 7 (3) ◽

pp. 57

Author(s):

Toyotaka Yoshida ◽

Yoshimasa Asano ◽

Kumiko Ui-Tei

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mirna Biogenesis ◽

Dependent Manner ◽

Rnase Iii ◽

Loop Region ◽

Dsrna Binding ◽

Essential Components ◽

Microrna Processing

MicroRNAs (miRNAs) are small non-coding RNAs that are about 22 nucleotides in length. They regulate gene expression post-transcriptionally by guiding the effector protein Argonaute to its target mRNA in a sequence-dependent manner, causing the translational repression and destabilization of the target mRNAs. Both Drosha and Dicer, members of the RNase III family proteins, are essential components in the canonical miRNA biogenesis pathway. miRNA is transcribed into primary-miRNA (pri-miRNA) from genomic DNA. Drosha then cleaves the flanking regions of pri-miRNA into precursor-miRNA (pre-miRNA), while Dicer cleaves the loop region of the pre-miRNA to form a miRNA duplex. Although the role of Drosha and Dicer in miRNA maturation is well known, the modulation processes that are important for regulating the downstream gene network are not fully understood. In this review, we summarized and discussed current reports on miRNA biogenesis caused by Drosha and Dicer. We also discussed the modulation mechanisms regulated by double-stranded RNA binding proteins (dsRBPs) and the function and substrate specificity of dsRBPs, including the TAR RNA binding protein (TRBP) and the adenosine deaminase acting on RNA (ADAR).

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C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons

Human Molecular Genetics ◽

10.1093/hmg/ddab089 ◽

2021 ◽

Author(s):

Eun Seon Kim ◽

Chang Geon Chung ◽

Jeong Hyang Park ◽

Byung Su Ko ◽

Sung Soon Park ◽

...

Keyword(s):

Retinal Degeneration ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Accumulation ◽

Heterozygous Mutation ◽

Pathological Feature ◽

Dependent Manner ◽

Cellular Processes ◽

Drosophila Model ◽

Post Transcriptional Regulation

Abstract RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS—but not with mutated endogenous NLS—potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/−), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/− is protective. Stau+/− also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

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RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton

Cytoskeleton ◽

10.1002/cm.21362 ◽

2017 ◽

Vol 74 (4) ◽

pp. 161-169 ◽

Cited By ~ 9

Author(s):

L. A. Mamon ◽

V. R. Ginanova ◽

S. F. Kliver ◽

A. O. Yakimova ◽

A. A. Atsapkina ◽

...

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins

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Matrin3: Disorder and ALS Pathogenesis

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.794646 ◽

2022 ◽

Vol 8 ◽

Author(s):

Ahmed Salem ◽

Carter J. Wilson ◽

Benjamin S. Rutledge ◽

Allison Dilliott ◽

Sali Farhan ◽

...

Keyword(s):

Nuclear Export ◽

Binding Proteins ◽

Rna Binding ◽

Nuclear Dna ◽

Neurodegenerative Disorder ◽

Rna Binding Proteins ◽

Binding Protein ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

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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Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains

Biological Chemistry ◽

10.1515/hsz-2013-0111 ◽

2013 ◽

Vol 394 (8) ◽

pp. 1077-1090 ◽

Cited By ~ 45

Author(s):

Kristin Wächter ◽

Marcel Köhn ◽

Nadine Stöhr ◽

Stefan Hüttelmaier

Keyword(s):

Subcellular Localization ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Accumulation ◽

Structural Constraints ◽

Cellular Functions ◽

Dependent Protein ◽

Mrna Binding

Abstract The IGF2 mRNA-binding protein family (IGF2BPs) directs the cytoplasmic fate of various target mRNAs and controls essential cellular functions. The three IGF2BP paralogues expressed in mammals comprise two RNA-recognition motifs (RRM) as well as four KH domains. How these domains direct IGF2BP paralogue-dependent protein function remains largely elusive. In this study, we analyze the role of KH domains in IGF2BPs by the mutational GXXG-GEEG conversion of single KH domain loops in the context of full-length polypeptides. These analyses reveal that all four KH domains of IGF2BP1 and IGF2BP2 are essentially involved in RNA-binding in vitro and the cellular association with RNA-binding proteins (RBPs). Moreover the KH domains prevent the nuclear accumulation of these two paralogues and facilitate their recruitment to stress granules. The role of KH domains appears less pronounced in IGF2BP3, because GxxG-GEEG conversion in all four KH domains only modestly affects RNA-binding, subcellular localization and RNA-dependent protein association of this paralogue. These findings indicate paralogue-dependent RNA-binding properties of IGF2BPs which likely direct distinct cellular functions. Our findings suggest that IGF2BPs contact target RNAs via all four KH domains. This implies significant structural constraints, which presumably allow the formation of exceedingly stable protein-RNA complexes.

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RNA Binding-Proteins Interact Specifically with the Arabidopsis Chloroplast psbA mRNA 5' Untranslated Region in a Redox-Dependent Manner

Plant and Cell Physiology ◽

10.1093/pcp/pce142 ◽

2001 ◽

Vol 42 (10) ◽

pp. 1071-1078 ◽

Cited By ~ 36

Author(s):

Y. Shen

Keyword(s):

Untranslated Region ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Dependent Manner

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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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RNA-binding protein DUS16 plays an essential role in primary miRNA processing in the unicellular alga Chlamydomonas reinhardtii

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1523230113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10720-10725 ◽

Cited By ~ 10

Author(s):

Tomohito Yamasaki ◽

Masayuki Onishi ◽

Eun-Jeong Kim ◽

Heriberto Cerutti ◽

Takeshi Ohama

Keyword(s):

Chlamydomonas Reinhardtii ◽

Essential Role ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Mature Mirnas ◽

Mirna Biogenesis ◽

Dsrna Binding

Canonical microRNAs (miRNAs) are embedded in duplexed stem–loops in long precursor transcripts and are excised by sequential cleavage by DICER nuclease(s). In this miRNA biogenesis pathway, dsRNA-binding proteins play important roles in animals and plants by assisting DICER. However, these RNA-binding proteins are poorly characterized in unicellular organisms. Here we report that a unique RNA-binding protein, Dull slicer-16 (DUS16), plays an essential role in processing of primary-miRNA (pri-miRNA) transcripts in the unicellular green alga Chlamydomonas reinhardtii. In animals and plants, dsRNA-binding proteins involved in miRNA biogenesis harbor two or three dsRNA-binding domains (dsRBDs), whereas DUS16 contains one dsRBD and also an ssRNA-binding domain (RRM). The null mutant of DUS16 showed a drastic reduction in most miRNA species. Production of these miRNAs was complemented by expression of full-length DUS16, but the expression of RRM- or dsRBD-truncated DUS16 did not restore miRNA production. Furthermore, DUS16 is predominantly localized to the nucleus and associated with nascent (unspliced form) pri-miRNAs and the DICER-LIKE 3 protein. These results suggest that DUS16 recognizes pri-miRNA transcripts cotranscriptionally and promotes their processing into mature miRNAs as a component of a microprocessor complex. We propose that DUS16 is an essential factor for miRNA production in Chlamydomonas and, because DUS16 is functionally similar to the dsRNA-binding proteins involved in miRNA biogenesis in animals and land plants, our report provides insight into this mechanism in unicellular eukaryotes.

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