The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

Ullman, Katharine S.; Shah, Sundeep; Powers, Maureen A.; Forbes, Douglass J.

doi:10.1091/mbc.10.3.649

Cited by 134 publications

(125 citation statements)

References 95 publications

(119 reference statements)

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“…In contrast, the Ftz and ␤-globin mRNAs used in this study are exported irrespective of prior splicing in vivo. Our export kinetics for DHFR mRNA are also more rapid than those reported by Lou and Reed (14), but are in agreement with those reported by several other laboratories (16,(23)(24)(25)(26). These results indicate that some cDNAderived mRNAs can be recognized as export substrates in Xenopus oocytes and can be exported efficiently.…”

Section: Function Of Conserved Domains Within Ref Proteinssupporting

confidence: 92%

REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus

Rodrigues¹

2001

Proceedings of the National Academy of Sciences

146

221

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The REF family of evolutionarily conserved heterogeneous ribonucleoprotein (hnRNP)-like proteins consists of one central RNPtype RNA binding domain flanked by Arg-Gly-rich regions of variable length. Members of this protein family bind directly to RNA and the mRNA export factor TAP͞Mex67p, and it has been suggested that they facilitate the recruitment of TAP͞Mex67p to cellular mRNPs. We show that the variable regions are necessary for binding of REFs to RNA and to TAP. Antibodies specific to REFs prevent their interaction with RNA in vitro. After microinjection into Xenopus oocytes, these antibodies inhibit mRNA nuclear export. This inhibition of export is observed whether or not the mRNAs are generated by splicing. The antibodies do not interfere with pre-mRNA splicing or with the nuclear export of constitutive transport element (CTE)-containing RNAs (directly mediated by TAP), so REF proteins must play a critical role in mRNA nuclear export, acting downstream of splicing and upstream of TAP͞ Mex67p. We also show that recombinant REFs stimulate directly the export of mRNAs that are otherwise exported inefficiently. Together, our data indicate that REFs are directly implicated in the export of mRNAs from the nucleus. More generally, we show that spliced and unspliced mRNAs use common export factors to reach the cytoplasm. Yra1p, a Saccharomyces cerevisiae member of the REF family, is an essential nuclear protein first identified from its RNAannealing activity (3). More recently, Yra1p was shown to be involved in the export of mRNA from the nucleus in yeast cells (2, 4). In mouse, REFs are encoded by at least three different genes (ref 1-3) and differ at multiple positions in the variable regions because of deletions and͞or amino acid changes (2). In contrast, all murine REF proteins are 98% identical in the RBD and 100% in the conserved boxes (2). The complexity of the murine subfamily is further increased by the expression of multiple splice variants (2). Murine REF1-II is generated by alternative splicing of REF1-I (also named Aly; see ref. 5) and lacks the N-terminal variable region (see Fig. 2 A), whereas murine REF2-I and REF2-II differ by one single amino acid insertion in REF2-I (Q198, ref. 2). REF1-I (Aly) was first identified as a protein interacting with LEF-1, a transcription factor that participates in the regulation of the T-cell receptor ␣ enhancer (5). In this context, it was proposed that Aly facilitates the interaction of multiple proteins in the T-cell receptor ␣ enhancer complex. In this study, we have defined the domains of REF interacting with RNA and TAP, and we have investigated the role of vertebrate REFs on the export of mRNAs from the nucleus. We show that the variable regions are required for binding of REFs to RNA and TAP. Antibodies specific to REFs, which prevent their binding to RNA in vitro, inhibit mRNA nuclear export when injected into Xenopus laevis oocytes. This export inhibition is observed whether or not the mRNAs have been generated by splicing. We show that microinjection ...

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Section: Function Of Conserved Domains Within Ref Proteinssupporting

confidence: 92%

REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus

Rodrigues¹

2001

Proceedings of the National Academy of Sciences

146

221

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“…4). Nup153 is associated with the basket structure on the nucleoplasmic side of the pore (19), and it has been implicated in multiple aspects of nuclear transport and pore structure (17,23). The association between Nup153 and SENP2 is therefore entirely consistent with immunofluorescence data showing that SENP2 was found only on the nucleoplasmic side of the nuclear envelope (Fig.…”

Section: Senp2 Targeting Regulates Its Activity Against Cellularsupporting

confidence: 83%

Association of the Human SUMO-1 Protease SENP2 with the Nuclear Pore

Hang

Dasso

2002

Journal of Biological Chemistry

221

184

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SUMO-1 is a small ubiquitin-like protein that can be covalently conjugated to other proteins. A family of proteases catalyzes deconjugation of SUMO-1-containing species. Members of this family also process newly synthesized SUMO-1 into its conjugatable form. To understand these enzymes better, we have examined the localization and behavior of the human SUMO-1 protease SENP2. Here we have shown that SENP2 associates with the nuclear face of nuclear pores and that this association requires protein sequences near the N terminus of SENP2. We have also shown that SENP2 binds to Nup153, a nucleoporin that is localized to the nucleoplasmic face of the pore. Nup153 binding requires the same domain of SENP2 that mediates its targeting in vivo. Removal of the Nup153-interacting region of SENP2 results in a significant change in the spectrum of SUMO-1 conjugates within the cell. Our results suggest that association with the pore plays an important negative role in the regulation of SENP2, perhaps by restricting its activity to a subset of the conjugated proteins within the nucleus. SUMO-11 is a ubiquitin-like protein that can be covalently conjugated to other proteins through an isopeptide linkage in a manner similar to ubiquitin (1). The SUMO-1 conjugation pathway utilizes proteins that both show sequence similarity to analogous enzymes in the ubiquitin pathway and utilize similar biochemical mechanisms (1). A large and growing number of SUMO-1 conjugation substrates have been reported in vertebrates (1). Notably, the profile of SUMO-1-conjugated proteins changes substantially in response to altered cellular conditions (see Ref. 2), suggesting that there are mechanisms to control the specificity of conjugation and/or deconjugation of SUMO-1 differentially between distinct substrates.Unlike enzymes of the SUMO-1 conjugation pathway, enzymes involved in SUMO processing and deconjugation are not closely related by sequence to their ubiquitin counterparts. Rather, known SUMO proteases share sequence homology in their catalytic domains, which is more nearly conserved to viral proteases (3). Two SUMO proteases have been described in budding yeast, Ulp1p (3) and Ulp2p/Smt4p (4, 5). Ulp1p is concentrated near the nuclear periphery (5) and interacts with nuclear pore components in two-hybrid assays (6), while Ulp2p is localized throughout the nucleus (5). ULP1 is an essential gene, and temperature-sensitive (ts) Ulp1p mutants arrest at the G2/M transition of the cell cycle. Ulp2 is not essential, but it is required for normal meiotic development, for regulation of spindle checkpoint arrest, and for chromatin condensation of rDNA during mitosis (4, 5). Interestingly, these proteins do not appear to act in a simple complementary manner, since ulp1-ts/ulp2-null double mutants grow better than ulp2 single mutants under a variety of conditions (5).In mammals, data base searches find at least seven members of the SUMO protease family (7), some of which have now been confirmed to act as SUMO proteases in vitro (8 -11). Outside of their c...

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“…Nup153 is a well-studied FG-nucleoporin, which is known to be critical for both nuclear import and nuclear export (Shah et al 1998;Ullman et al 1999; for review see Ball and Ullman 2005). Immuno-EM studies of the vertebrate Nup153 have clearly demonstrated a high degree of mobility and structural flexibility of its FG-repeat domain .…”

Section: Nucleocytoplasmic Transport Across the Nuclear Pore Complex:mentioning

confidence: 99%

From the trap to the basket: getting to the bottom of the nuclear pore complex

2006

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Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the doublemembraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of highresolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed 'minimalist' NPC.

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The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

Cited by 134 publications

References 95 publications

REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus

REF proteins mediate the export of spliced and unspliced mRNAs from the nucleus

Association of the Human SUMO-1 Protease SENP2 with the Nuclear Pore

From the trap to the basket: getting to the bottom of the nuclear pore complex

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