Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.     

长链非编码RNA通过细胞核高级结构调控真核基因表达及其临床意义

长链非编码RNA(long non-coding RNA, lncRNA)是一类转录本长度超过200nt、不编码蛋白质的RNA。近年来,随着染色质构象捕获及转录组测序等技术的发展,lncRNA与染色质构象间的关系越来越受到重视。多项研究表明,lncRNA在基因调控网络中具有重要的作用,可通过影响细胞核高级结构的动态变化来调控真核基因的表达。因其广泛的基因调控功能及在肿瘤发生过程中的重要作用,lncRNA被认为是未来肿瘤临床诊断和预后判定的新型标志物之一。本文旨在介绍lncRNA改变细胞核高级结构从而调控关键基因表达的分子机制,并详细介绍lncRNA在肿瘤治疗中的临床意义。     

CCCTC-结合因子与核周蛋白α4存在分子间相互作用并调控其基因表达

目的:研究CCCTC-结合因子(CTCF)是否与核周蛋白α4(KPNA4)有蛋白间相互作用并调控其表达。方法:用GSTpull-down实验研究CTCF是否与KPNA4存在蛋白间相互作用;用逆转录半定量PCR和免疫印迹检测CTCF敲降对KPNA4基因表达的影响。结果:GSTpull-down实验表明CTCF与KPNA4之间存在相互作用;当CTCF敲降时,KPNA4基因的表达随之下降。结论:CTCF与KPNA4之间存在相互作用并调控其基因表达。     

Gradient of increasing affinity of importin beta for nucleoporins along the pathway of nuclear import

Nuclear import and export signals on macromolecules mediate directional, receptor-driven transport through the nuclear pore complex (NPC) by a process that is suggested to involve the sequential binding of transport complexes to different nucleoporins. The directionality of transport appears to be partly determined by the nucleocytoplasmic compartmentalization of components of the Ran GTPase system. We have analyzed whether the asymmetric localization of discrete nucleoporins can also contribute to transport directionality. To this end, we have used quantitative solid phase binding analysis to determine the affinity of an importin beta cargo complex for Nup358, the Nup62 complex, and Nup153, which are in the cytoplasmic, central, and nucleoplasmic regions of the NPC, respectively. These nucleoporins are proposed to provide progressively more distal binding sites for importin beta during import. Our results indicate that the importin beta transport complex binds to nucleoporins with progressively increasing affinity as the complex moves from Nup358 to the Nup62 complex and to Nup153. Antibody inhibition studies support the possibility that importin beta moves from Nup358 to Nup153 via the Nup62 complex during import. These results indicate that nucleoporins themselves, as well as the nucleocytoplasmic compartmentalization of the Ran system, are likely to play an important role in conferring directionality to nuclear protein import.     

Nup2p dynamically associates with the distal regions of the yeast nuclear pore complex.

Nucleocytoplasmic transport is mediated by the interplay between soluble transport factors and nucleoporins resident within the nuclear pore complex (NPC). Understanding this process demands knowledge of components of both the soluble and stationary phases and the interface between them. Here, we provide evidence that Nup2p, previously considered to be a typical yeast nucleoporin that binds import- and export-bound karyopherins, dynamically associates with the NPC in a Ran-facilitated manner. When bound to the NPC, Nup2p associates with regions corresponding to the nuclear basket and cytoplasmic fibrils. On the nucleoplasmic face, where the Ran--GTP levels are predicted to be high, Nup2p binds to Nup60p. Deletion of NUP60 renders Nup2p nucleoplasmic and compromises Nup2p-mediated recycling of Kap60p/Srp1p. Depletion of Ran--GTP by metabolic poisoning, disruption of the Ran cycle, or in vitro by cell lysis, results in a shift of Nup2p from the nucleoplasm to the cytoplasmic face of the NPC. This mobility of Nup2p was also detected using heterokaryons where, unlike nucleoporins, Nup2p was observed to move from one nucleus to the other. Together, our data support a model in which Nup2p movement facilitates the transition between the import and export phases of nucleocytoplasmic transport.     

A synthetic peptide bearing the HIV-1 integrase 161-173 amino acid residues mediates active nuclear import and binding to importin alpha: characterization of a functional nuclear localization signal

In spite of recent efforts to elucidate the nuclear import pathway of the human immunodeficiency virus type 1 (HIV-1) integrase protein (IN), its exact route as well as the domains that mediate its import are still unknown. Here, we show that a synthetic peptide bearing the amino acid residues 161-173 of the HIV-1 IN is able to mediate active import of covalently attached bovine serum albumin molecules into nuclei of permeabilized cells and therefore was designated as nuclear localization signal-IN (NLS(IN)). A peptide bearing residues 161-173 in the reversed order showed low karyophilic properties. Active nuclear import was demonstrated by using fluorescence microscopy and a quantitative ELISA-based assay system. Nuclear import was blocked by addition of the NLS(IN) peptide, as well as by a peptide bearing the NLS of the simian virus 40 T-antigen (NLS-SV40). The NLS(IN) peptide partially inhibited nuclear import mediated by the full-length recombinant HIV-1 IN protein, indicating that the sequence of the NLS(IN) is involved in mediating nuclear import of the IN protein. The NLS(IN) as well as the full-length IN protein interacted specifically with importin alpha, binding of which was blocked by the NLS(IN) peptide itself as well as by the NLS-SV40.     

Human ribosomal protein S13 inhibits splicing of its own pre-mRNA

Recombinant human ribosomal protein (rp) S13 was shown to specifically bind with its own pre-mRNA fragment containing the first exon, first intron, second exon, and a part of the second intron and to inhibit its splicing in vitro. The binding of rpS13 was specific: recombinant human rpS10 and rpS16 bound with the fragment to a lower extent. Moreover, rpS13 binding with the rpS13 pre-mRNA fragment was inhibited by non-labeled poly(AU) and an adenovirus pre-mRNA fragment to a lower extent than by the nonlabeled rpS13 pre-mRNA fragment. The specificity of splicing inhibition was inferred from the finding that, in contrast to rpS13, recombinant rpS10 and rpS16 did not affect the efficiency of first intron excision from the rpS13 pre-mRNA fragment. Enzymatic footprinting was used to determine the rpS13 pre-mRNA nucleotides whose accessibility to RNases T1, T2, and V1 changed in the presence of rpS13. Such nucleotides were detected close to the 5′ and 3′ splicing sites of the first intron. Analysis with the EMBOS-Align program showed that the nucleotide sequence of the first intron of the mammalian rpS13 pre-mRNA is conserved to a greater extent as compared with the other introns. It was assumed that the first intron plays an important role in regulating the expression of the rpS13 gene at the splicing level in all mammals.     

Nuclear transport of Kir/Gem requires specific signals and importin alpha5 and is regulated by calmodulin and predicted serine phosphorylations

Kir/Gem, together with Rad, Rem and Rem2, is a member of the RGK small GTP-binding protein family. These multifunctional proteins regulate voltage-gated calcium channel (VGCC) activity and cell-shape remodeling. Calmodulin and 14-3-3 binding modulate the functions of RGK proteins. Intriguingly, abolishing the binding of calmodulin or calmodulin and 14-3-3 results in nuclear accumulation of RGK proteins. Under certain conditions, the Ca(v)beta3-subunit of VGCCs can be translocated into the nucleus along with the RGK proteins, resulting in channel inactivation. The mechanism by which nuclear localization of RGK proteins is accomplished and regulated, however, is unknown. Here, we identify specific nuclear localization signals (NLS) in Kir/Gem that are both required and sufficient for nuclear transport. Importin alpha5 binds to Kir/Gem, and its depletion using RNA interference impairs nuclear translocation of this RGK protein. Calmodulin and predicted phosphorylations on serine residues within or in the vicinity of a C-terminal bipartite NLS regulate nuclear translocation by interfering with the association between importinalpha5 and Kir/Gem. These predicted phosphorylations, however, do not affect Kir/Gem-mediated calcium channel downregulation but rather, as shown in the accompanying paper (Mahalakshmi RN, Ng MY, Guo K, Qi Z, Hunziker W, Béguin P. Nuclear localization of endogenous RGK proteins and modulation of cell shape remodeling by regulated nuclear transport. Traffic 2007; doi:10.1111/j.1600-0854.2007.00599.x), interfere with cell-shape remodeling.