Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export

Abstract

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.

The Entire Nup107-160 Complex, Including Three New Members, Is Targeted as One Entity to Kinetochores in Mitosis

“…A faint signal could also be observed at the NE in both live and fixed interphase HeLa cells (Figure 8, A and B). This dual ER/NE localization is in agreement with the biochemical interaction of Sec13 with both Sec31, a constituent of the COPII complex (Tang et al, 2000; see also Figure 3), and the various constituents of the Nup107-160 NPC subcomplex (Vasu et al, 2001;Enninga et al, 2003; see also Figure 3). In addition, a very faint signal also could be observed at kinetochores in live mitotic cells (Figure 8A, inset).…”

“…To further characterize the behavior of these nucleoporins, positive fractions from the sucrose gradient were analyzed by gel filtration chromatography. As shown in Figure 2B, Nup133, Nup85, and Nup37 were all eluted much before Thyroglobulin (670 kDa) on a Superose 6 column ( Figure 2B), a result consistent with the behavior of the Xenopus Nup107-160 complex upon fractionation on Sephacryl S400-HR column (Vasu et al, 2001). The unexpected behavior of the Nup107-160 complex on sucrose gradient may reflect its elongated shape, similar to the previously described Y-shaped structure of the S. cerevisiae Nup84 complex .…”

“…In addition, we noticed a cytoplasmic accumulation of SC-35 foci that correlated with a minor increase of G1-like cells in some Nup133-, Nup107- (Walther et al, 2003a; our unpublished data), Seh1-, but also Nup153-depleted cells. These common late phenotypes might thus reflect the interaction between Nup153 and the Nup107-160 complex (Vasu et al, 2001) and/or their common involvement in NPC assembly at the end of mitosis (Walther et al, 2003b). Alternatively, and as recently described in the case of gp210 depletion, these late phenotypes could be less specific and reflect a population of sub-G1 cells at an early stage of apoptosis (Cohen et al, 2003).…”

“…Constituents of this complex are localized on both sides of the NPCs in yeast (Rout et al, 2000) and vertebrates (Belgareh et al, 2001;Enninga et al, 2003). This complex was shown to be involved in NPC distribution in S. cerevisiae, and in mRNA export both in yeast (reviewed in Doye and Hurt, 1997) and vertebrates (Vasu et al, 2001;Boehmer et al, 2003;Walther et al, 2003a). More recently, this complex was demonstrated to also be critical for NPC assembly in vertebrates (Harel et al, 2003;Walther et al, 2003a).…”

“…Isolation under native conditions and in vitro reconstitution have further revealed the Y-shaped structure of this complex (Siniossoglou et al, 2000;Lutzmann et al, 2002). The vertebrate Nup107-160 complex was so far demonstrated to consist of Nup107 (homologous to ScNup84), Nup133 (homologous to ScNup133), Nup160 (homologous to ScNup120 and thus also referred in human cells as Nup120), Nup96 (homologous to ScNup145-C), Sec13 and Nup85/75 (Fontoura et al, 1999;Belgareh et al, 2001;Vasu et al, 2001;Harel et al, 2003). In addition, previous studies reported the cofractionation on sucrose gradients of Nup107, Nup96, and Sec13 together with a 37-kDa WD-repeat containing protein (Fontoura et al, 1999), subsequently identified as Nup37 (Cronshaw et al, 2002).…”