Nuclear RNA binding regulates TDP-43 nuclear localization and passive nuclear export

AbstractNuclear clearance of the DNA/RNA-binding protein TDP-43 is a pathologic hallmark of amyotrophic lateral sclerosis and frontotemporal dementia that remains unexplained. Moreover, our current understanding of TDP-43 nucleocytoplasmic shuttling does not fully explain the predominantly nuclear localization of TDP-43 in healthy cells. Here, we used permeabilized and live-cell models to investigate TDP-43 nuclear export and the role of RNA in TDP-43 localization. We show that TDP-43 nuclear efflux occurs in low-ATP conditions and independent of active mRNA export, consistent with export by passive diffusion through nuclear pore channels. TDP-43 nuclear residence requires binding to GU-rich nuclear intronic pre-mRNAs, based on the induction of TDP-43 nuclear efflux by RNase and GU-rich oligomers and TDP-43 nuclear retention conferred by pre-mRNA splicing inhibitors. Mutation of TDP-43 RNA recognition motifs disrupts TDP-43 nuclear accumulation and abolishes transcriptional blockade-induced TDP-43 nuclear efflux, demonstrating strict dependence of TDP-43 nuclear localization on RNA binding. Thus, the nuclear abundance of GU-rich intronic pre-mRNAs, as dictated by the balance of transcription and pre-mRNA processing, regulates TDP-43 nuclear sequestration and availability for passive nuclear export.

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Domain necessary for Drosophila ELAV nuclear localization: function requires nuclear ELAV

Journal of Cell Science ◽

10.1242/jcs.112.24.4501 ◽

1999 ◽

Vol 112 (24) ◽

pp. 4501-4512 ◽

Cited By ~ 1

Author(s):

Y.M. Yannoni ◽

K. White

Keyword(s):

Nuclear Localization ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Localization Sequence ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Mutant Proteins ◽

Localization Sequence ◽

Recognition Motifs

The neuron specific Drosophila ELAV protein belongs to the ELAV family of RNA binding proteins which are characterized by three highly conserved RNA recognition motifs, an N-terminal domain, and a hinge region between the second and third RNA recognition motifs. Despite their highly conserved RNA recognition motifs the ELAV family members are a group of proteins with diverse posttranscriptional functions including splicing regulation, mRNA stability and translatability and have a variety of subcellular localizations. The role of the ELAV hinge in localization and function was examined using transgenes encoding ELAV hinge deletions, in vivo. Subcellular localization of the hinge mutant proteins revealed that residues between amino acids 333–374 are necessary for nuclear localization. This delineated sequence has no significant homology to classical nuclear localization sequences, but it is similar to the recently characterized nucleocytoplasmic shuttling sequence, the HNS, from a human ELAV family member, HuR. This defined sequence, however, was insufficient for nuclear localization as tested using hinge-GFP fusion proteins. Functional assays revealed that mutant proteins that fail to localize to the nucleus are unable to provide ELAV vital function, but their function is significantly restored when translocated into the nucleus by a heterologous nuclear localization sequence tag.

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CaMKII and PKA-dependent phosphorylation co-regulate nuclear localization of HDAC4 in adult cardiomyocytes

Basic Research in Cardiology ◽

10.1007/s00395-021-00850-2 ◽

2021 ◽

Vol 116 (1) ◽

Author(s):

Kathryn G. Helmstadter ◽

Senka Ljubojevic-Holzer ◽

Brent M. Wood ◽

Khanha D. Taheri ◽

Simon Sedej ◽

...

Keyword(s):

Nuclear Localization ◽

Nuclear Export ◽

Ventricular Myocytes ◽

Early Response ◽

Confocal Imaging ◽

Nuclear Accumulation ◽

Nuclear Retention ◽

Human Cardiomyocytes ◽

Mediated Effects ◽

In Cells

AbstractNuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII—but not PKA, PKC or PKD—raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (FNuc/FCyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca2+], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising FNuc/FCyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

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Nuclear transit of human zipcode-binding protein IMP1

Biochemical Journal ◽

10.1042/bj20030943 ◽

2003 ◽

Vol 376 (2) ◽

pp. 383-391 ◽

Cited By ~ 27

Author(s):

Jacob NIELSEN ◽

Sidsel K. ADOLPH ◽

Ewa RAJPERT-De MEYTS ◽

Jens LYKKE-ANDERSEN ◽

Grete KOCH ◽

...

Keyword(s):

Translational Control ◽

Nuclear Export ◽

Rna Binding ◽

Binding Protein ◽

Leptomycin B ◽

Granule Formation ◽

Rna Recognition Motifs ◽

3T3 Fibroblasts ◽

Recognition Motifs ◽

Homology Domains

The human IMPs (insulin-like growth factor II mRNA-binding proteins) belong to a vertebrate zipcode-binding protein family consisting of two RNA recognition motifs and four K homology domains and have been implicated in cytoplasmic mRNA localization, turnover and translational control. In the present study, we show that IMP1 is capable of translocating into nuclei of NIH 3T3 fibroblasts and its immunoreactivity is present in the nuclei of human spermatogenic cells. IMP1 does not contain a simple import signal, but nuclear entry was facilitated by disruption of RNA binding and cytoplasmic granule formation. IMP1 contains two NESs (nuclear export signals) within the RNA-binding K homology domains 2 and 4. The former is a leucine-rich leptomycin B-sensitive NES, whereas the latter is a leptomycin B-insensitive NES. Taken together, these results indicate that IMP1 may attach to its target mRNAs in the nucleus and thereby define the cytoplasmic fate of the transcripts.

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The RNA-Binding Motif Protein Family in Cancer: Friend or Foe?

Frontiers in Oncology ◽

10.3389/fonc.2021.757135 ◽

2021 ◽

Vol 11 ◽

Author(s):

Zhigang Li ◽

Qingyu Guo ◽

Jiaxin Zhang ◽

Zitong Fu ◽

Yifei Wang ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Binding Motif ◽

Rna Recognition ◽

Aberrant Expression ◽

Rna Recognition Motifs ◽

Binding Domains ◽

Cancer Occurrence ◽

Recognition Motifs

The RNA-binding motif (RBM) proteins are a class of RNA-binding proteins named, containing RNA-recognition motifs (RRMs), RNA-binding domains, and ribonucleoprotein motifs. RBM proteins are involved in RNA metabolism, including splicing, transport, translation, and stability. Many studies have found that aberrant expression and dysregulated function of RBM proteins family members are closely related to the occurrence and development of cancers. This review summarizes the role of RBM proteins family genes in cancers, including their roles in cancer occurrence and cell proliferation, migration, and apoptosis. It is essential to understand the mechanisms of these proteins in tumorigenesis and development, and to identify new therapeutic targets and prognostic markers.

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Nuclear Export and Retention Signals in the RS Domain of SR Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.22.19.6871-6882.2002 ◽

2002 ◽

Vol 22 (19) ◽

pp. 6871-6882 ◽

Cited By ~ 103

Author(s):

Demian Cazalla ◽

Jun Zhu ◽

Lisa Manche ◽

Elisabeth Huber ◽

Adrian R. Krainer ◽

...

Keyword(s):

Subcellular Localization ◽

Protein Interactions ◽

Nuclear Export ◽

Sr Proteins ◽

Nucleocytoplasmic Shuttling ◽

Sr Protein ◽

Nuclear Retention ◽

Rna Recognition Motifs ◽

Recognition Motifs

ABSTRACT Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and—in certain situations—nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.

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Nucleocytoplasmic Distribution of Human RNA-editing Enzyme ADAR1 Is Modulated by Double-stranded RNA-binding Domains, a Leucine-rich Export Signal, and a Putative Dimerization Domain

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0161 ◽

2002 ◽

Vol 13 (11) ◽

pp. 3822-3835 ◽

Cited By ~ 67

Author(s):

Alexander Strehblow ◽

Martina Hallegger ◽

Michael F. Jantsch

Keyword(s):

Rna Editing ◽

Nuclear Localization ◽

Nuclear Export ◽

Rna Binding ◽

Nuclear Export Signal ◽

Nuclear Accumulation ◽

Double Stranded Rna ◽

Cytoplasmic Accumulation ◽

Editing Enzyme ◽

Export Signal

The human RNA-editing enzyme adenosine deaminase that acts on RNA (ADAR1) is expressed in two versions. A longer 150-kDa protein is interferon inducible and can be found both in the nucleus and cytoplasm. An amino-terminally truncated 110-kDa version, in contrast, is constitutively expressed and predominantly nuclear. In the absence of transcription, however, the shorter protein is also cytoplasmic and thus displays the hallmarks of a shuttling protein. The nuclear localization signal (NLS) of human hsADAR1 is atypical and overlaps with its third double-stranded RNA-binding domain (dsRBD). Herein, we identify regions in hsADAR1 that interfere with nuclear localization and mediate cytoplasmic accumulation. We show that interferon-inducible hsADAR1 contains a Crm1-dependent nuclear export signal in its amino terminus. Most importantly, we demonstrate that the first dsRBD of hsADAR1 interferes with nuclear localization of a reporter construct containing dsRBD3 as an active NLS. The same effect can be triggered by several other, but not all dsRBDs. Active RNA binding of either the inhibitory dsRBD1 or the NLS bearing dsRBD3 is required for cytoplasmic accumulation. Furthermore, hsADAR1's dsRBD1 has no effect on other NLSs, suggesting RNA-mediated cross talk between dsRBDs, possibly leading to masking of the NLS. A model, incorporating these findings is presented. Finally, we identify a third region located in the C terminus of hsADAR1 that also interferes with nuclear accumulation of this protein.

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Conservation of a Masked Nuclear Export Activity of La Proteins and Its Effects on tRNA Maturation

Molecular and Cellular Biology ◽

10.1128/mcb.00026-07 ◽

2007 ◽

Vol 27 (9) ◽

pp. 3303-3312 ◽

Cited By ~ 25

Author(s):

Mark A. Bayfield ◽

Trish E. Kaiser ◽

Robert V. Intine ◽

Richard J. Maraia

Keyword(s):

Fission Yeast ◽

Nuclear Export ◽

Rna Binding ◽

Nuclear Accumulation ◽

Rna Recognition Motif ◽

Nuclear Retention ◽

Trna Processing ◽

Export Activity ◽

La Protein

ABSTRACT La is an RNA-processing-associated phosphoprotein so highly conserved that the human La protein (hLa) can replace the tRNA-processing function of the fission yeast La protein (Sla1p) in vivo. La proteins contain multiple trafficking elements that support interactions with RNAs in different subcellular locations. Prior data indicate that deletion of a nuclear retention element (NRE) causes nuclear export of La and dysfunctional processing of associated pre-tRNAs that are spliced but 5′ and 3′ unprocessed, with an accompanying decrease in tRNA-mediated suppression, in fission yeast. To further pursue these observations, we first identified conserved residues in the NREs of hLa and Sla1p that when substituted mimic the NRE deletion phenotype. NRE-defective La proteins then deleted of other motifs indicated that RNA recognition motif 1 (RRM1) is required for nuclear export. Mutations of conserved RRM1 residues restored nuclear accumulation of NRE-defective La proteins. Some RRM1 mutations restored nuclear accumulation, prevented disordered pre-tRNA processing, and restored suppression, indicating that the tRNA-related activity of RRM1 and its nuclear export activity could be functionally separated. When mapped onto an hLa structure, the export-sensitive residues comprised surfaces distinct from the RNA-binding surface of RRM1. The data indicate that the NRE has been conserved to mask or functionally override an equally conserved nuclear export activity of RRM1. The data suggest that conserved elements mediate nuclear retention, nuclear export, and RNA-binding activities of the multifunctional La protein and that their interrelationship contributes to the ability of La to engage its different classes of RNA ligands in different cellular locations.

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Faculty Opinions recommendation of The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1025155.298750 ◽

2005 ◽

Author(s):

Reinhard Lührmann

Keyword(s):

Rna Binding ◽

Splicing Factor ◽

Rna Recognition ◽

Rna Recognition Motifs ◽

Recognition Motifs ◽

U6 Rna

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Mex67p of Schizosaccharomyces pombeInteracts with Rae1p in Mediating mRNA Export

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8767-8782.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8767-8782 ◽

Cited By ~ 52

Author(s):

Jin Ho Yoon ◽

Dona C. Love ◽

Anjan Guhathakurta ◽

John A. Hanover ◽

Ravi Dhar

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Fluorescent Protein ◽

Synthetic Lethality ◽

Sequence Similarity ◽

Mrna Export ◽

Nuclear Periphery ◽

Nuclear Pore ◽

Multicopy Suppressor ◽

Accessory Factor

ABSTRACT We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 tsmutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast toscMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with therae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of therae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

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