Heterogeneous nuclear ribonucleoprotein B1 protein impairs DNA repair mediated through the inhibition of DNA-dependent protein kinase activity

Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.     

Molecular basis for dissimilar nuclear trafficking of the actin-bundling protein isoforms T- and L-plastin

T- and L-plastin are highly similar actin-bundling proteins implicated in the regulation of cell morphology, lamellipodium protrusion, bacterial invasion and tumor progression. We show that T-plastin localizes predominantly to the cytoplasm, whereas L-plastin distributes between nucleus and cytoplasm in HeLa or Cos cells. T-plastin shows nuclear accumulation upon incubation of cells with the CRM1 antagonist leptomycin B (LMB). We identified a Rev-like nuclear export sequence (NES) in T-plastin that is able to export an otherwise nuclear protein in an LMB-dependent manner. Deletion of the NES promotes nuclear accumulation of T-plastin. Mutation of residues L17, F21 or L26 in the T-plastin NES inhibits nuclear efflux. L-plastin harbors a less conserved NES and lacks the F21 T-plastin residue. Insertion of a Phe residue in the L-plastin NES specifically enhances its export activity. These findings explain why both isoforms exhibit specific distribution patterns in eukaryotic cells.     

RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.     

High‐resolution mapping of function and protein binding in an RNA nuclear enrichment sequence

The functions of long RNAs, including mRNAs and long noncoding RNAs (lncRNAs), critically depend on their subcellular localization. The identity of the sequences that dictate subcellular localization and their high‐resolution anatomy remain largely unknown. We used a suite of massively parallel RNA assays and libraries containing thousands of sequence variants to pinpoint the functional features within the SIRLOIN element, which dictates nuclear enrichment through hnRNPK recruitment. In addition, we profiled the endogenous SIRLOIN RNA‐nucleoprotein complex and identified the nuclear RNA‐binding proteins SLTM and SNRNP70 as novel SIRLOIN binders. Taken together, using massively parallel assays, we identified the features that dictate binding of hnRNPK, SLTM, and SNRNP70 to SIRLOIN and found that these factors are jointly required for SIRLOIN activity. Our study thus provides a roadmap for high‐throughput dissection of functional sequence elements in long RNAs.     

Systematic mapping of nuclear domain-associated transcripts reveals speckles and lamina as hubs of functionally distinct retained introns

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A nuclear matrix protein related to intermediate filaments proteins is a member of the complex binding alphoid DNA in vitro

A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins.     

Characterisation of nuclear pore complex oxalate binding protein from human kidney

Both rat and human kidney nuclei exhibited time and pH dependent oxalate or histone-oxalate uptake which was inhibited by anion transport inhibitor, 4,4-dithiocyanostilbene-2,2-disulphonic acid. Sodium chloride had no effect. Nuclear membrane had oxalate binding at pH 7.4. Extraction of nuclear membrane by Triton–high salt mixture showed maximal oxalate binding activity with nuclear pore complex while nuclear lamin had no oxalate binding. The rat and human kidney nuclear pore complex showed oxalate binding of 144 and 220 pmoles/mg protein respectively. Subsequent purification of the protein on diethyl amino ethyl–Sephadex A 50 column and Sephadex G-200 column yielded 4-fold purification. The protein revealed a molecular weight of 205 kDa on SDS-PAGE. The protein was found to be saturable at 2 M oxalate and had a Kd of 2.98 pM and a Bmax of 197 pmoles. Antibody for 205 kD was separated from primary biliary cirrhosis serum containing auto antibody against 205 kDa using affinity column chromatography. The oxalate binding activity as well as the nuclear uptake of oxalate or histone-oxalate were inhibited by its antibody.     

Effect of cisplatin treatment on speckled distribution of a serine/arginine-rich nuclear protein CROP/Luc7A

The C-half of cisplatin resistance-associated overexpressed protein (CROP), an SR-related protein, comprises domains rich in arginine and glutamate residues (RE domain), and is rich in arginine and serine residues (RS domain). We analyzed the role of the individual domains of CROP in cellular localization, subnuclear localization, and protein-protein interaction. CROP fused with green fluorescent protein, GFP-CROP, localized exclusively to the nucleus and showed a speckled intranuclear distribution. The yeast two-hybrid system revealed that CROP interacted with SF2/ASF, an SR protein involved in RNA splicing, as well as CROP itself. The RE and RS domains were necessary for both the intranuclear speckled distribution and the protein-protein interaction. CROP was phosphorylated by mSRPK1, mSRPK2, and Clk1 in vitro, and when cells were treated with cisplatin the subnuclear distribution of GFP-CROP was changed. These results suggest that cisplatin affects RNA splicing by changing the subnuclear distribution of SR proteins including CROP.     

Mitogen activated protein kinase at the nuclear pore complex

Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD‐98059, two MAP kinase antagonists as well as with SB‐202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N‐terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function.     

NUFIP1 (nuclear FMRP interacting protein 1) is a nucleocytoplasmic shuttling protein associated with active synaptoneurosomes

Fragile X syndrome, the most common cause of inherited mental retardation, is caused by the absence of FMRP (Fragile X Mental Retardation Protein). FMRP is an RNA binding protein reported to be involved in translational control, notably at postsynaptic sites of protein synthesis as a part of a multiprotein/mRNA complex. One of the FMRP interactors, NUFIP1, is an RNA binding protein with an expression profile matching that of FMRP. We now show that in the nucleus NUFIP1 is localized in the nuclear matrix in RNA-containing structures lying in the proximity of, but not overlapping with, sites of nascent RNA. NUFIP1 is also present in the cytoplasm, where it is associated with ribosomes, similarly to FMRP. In neurons NUFIP1 can be detected in functional synaptoneurosomes, colocalizing with ribosomes. Consistent with its subcellular localization in both nucleus and cytoplasm, we show that NUFIP1 contains a functional CRM1-dependent nuclear export signal and is able to shuttle between these two cellular compartments. These findings suggest the involvement of NUFIP1 in the export and localization of mRNA and, in association with FMRP, in the regulation of local protein synthesis near synapses.     

The Wilms tumor protein is persistently associated with the nuclear matrix throughout the cell cycle

In order to investigate the subnuclear interactions of the WTI gene product, nuclear fractionation analyses were performed with human osteosarcoma HOS and myelogenous leukemia K562 cells. The WT1 protein was tightly associated with the nucleus and was resistant to high-salt or derergent extraction and DNase I digestion. Both the expression level and stability of WT1 and its resistance to high salt and DNase I treatments remained constant during the cell cycle. In addition, human WT1 ectopically expressed in mouse NIH3T3 cells was also resistant to these treatments. These results suggest that WT1 functions in tight association with the nuclear matrix.     

Exclusion of m6A from splice-site proximal regions by the exon junction complex dictates m6A topologies and mRNA stability

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Cloning of a protein arginine methyltransferase PRMT1 homologue from Schistosoma mansoni: evidence for roles in nuclear receptor signaling and RNA metabolism

The most studied arginine methyltransferase is the type I enzyme, which catalyzes the transfer of an S-adenosyl-L-methionine to a broad spectrum of substrates, including histones, RNA-transporting proteins, and nuclear hormone receptor coactivators. We cloned a cDNA encoding a protein arginine methyltransferase in Schistosoma mansoni (SmPRMT1). SmPRMT1 is highly homologous to the vertebrate PRMT1 enzyme. In vitro methylation assays showed that SmPRMT1 recombinant protein was able to specifically methylate histone H4. Two schistosome proteins likely to be involved in RNA metabolism, SMYB1 and SmSmD3, that display a number of RGG motifs, were strongly methylated by SmPRMT1. In vitro GST pull-down assays showed that SMYB1 and SmSmD3 physically interacted with SmPRMT1. Additional GST pull-down assay suggested the occurrence of a ternary complex including SmPRMT1, SmRXR1 nuclear receptor, and the p160 (SRC-1) nuclear receptor coactivator. Together, these data suggest a mechanism by which SmPRMT1 plays a role in nuclear receptor-mediated chromatin remodeling and RNA transactions.     

In vitro reconstitution of yeast splicing with U4 snRNA reveals multiple roles for the 3' stem-loop

U4 small nuclear RNA (snRNA) plays a fundamental role in the process of premessenger RNA splicing, yet many questions remain regarding the location, interactions, and roles of its functional domains. To address some of these questions, we developed the first in vitro reconstitution system for yeast U4 small nuclear ribonucleoproteins (snRNPs). We used this system to examine the functional domains of U4 by measuring reconstitution of splicing, U4/U6 base-pairing, and triple-snRNP formation. In contrast to previous work in human extracts and Xenopus oocytes, we found that the 3' stem-loop of U4 is necessary for efficient base-pairing with U6. In particular, the loop is sensitive to changes in both length and sequence. Intriguingly, a number of mutations that we tested resulted in more stable interactions with U6 than wild-type U4. Nevertheless, each of these mutants was impaired in its ability to support splicing, indicating that these regions of U4 have functions subsequent to base pair formation with U6. Our data suggest that one such function is likely to be in tri-snRNP formation, when U5 joins the U4/U6 di-snRNP. We have identified two regions, the upper stem of the 3' stem-loop and the central domain, that promote tri-snRNP formation. In addition, the loop of the 3' stem-loop promotes di-snRNP formation, while the central domain and the 3'-terminal domain appear to antagonize di-snRNP formation.     

SUMOylation mediates the disassembly of the Smad4 nuclear export complex via RanGAP1 in KELOIDS

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-β/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.     

The exon junction complex component Y14 modulates the activity of the methylosome in biogenesis of spliceosomal small nuclear ribonucleoproteins

The RNA-binding protein Y14 heterodimerizes with Mago as the core of the exon junction complex during precursor mRNA splicing and plays a role in mRNA surveillance in the cytoplasm. Using the Y14/Magoh heterodimer as bait in a screening for its interacting partners, we identified the protein-arginine methyltransferase PRMT5 as a candidate. We show that Y14 and Magoh, but not other factors of the exon junction complex, interact with the cytoplasmic PRMT5-containing methylosome. We further provide evidence that Y14 promoted the activity of PRMT5 in methylation of Sm proteins of the small nuclear ribonucleoprotein core, whereas knockdown of Y14 reduced their methylation level. Moreover, Y14 overexpression induced the formation of a large, active, and small nuclear ribonucleoprotein (snRNP)-associated methylosome complex. However, Y14 may only transiently associate with the snRNP assembly complex in the cytoplasm. Together, our results suggest that Y14 facilitates Sm protein methylation probably by its activity in promoting the formation or stability of the methylosome-containing complex. We hypothesize that Y14 provides a regulatory link between pre-mRNA splicing and snRNP biogenesis.