Changes in the transcriptional state of genes have been correlated with their repositioning within the nuclear space. Tethering reporter genes to the nuclear envelope alone can impose repression and recent reports have shown that, after activation, certain genes can also be found closer to the nuclear periphery. The molecular mechanisms underlying these phenomena have remained elusive. Here, with the use of dynamic three-dimensional tracking of a single locus in live yeast (Saccharomyces cerevisiae) cells, we show that the activation of GAL genes (GAL7, GAL10 and GAL1) leads to a confinement in dynamic motility. We demonstrate that the GAL locus is subject to sub-diffusive movement, which after activation can become constrained to a two-dimensional sliding motion along the nuclear envelope. RNA-fluorescence in situ hybridization analysis after activation reveals a higher transcriptional activity for the peripherally constrained GAL genes than for loci remaining intranuclear. This confinement was mediated by Sus1 and Ada2, members of the SAGA histone acetyltransferase complex, and Sac3, a messenger RNA export factor, physically linking the activated GAL genes to the nuclear-pore-complex component Nup1. Deleting ADA2 or NUP1 abrogates perinuclear GAL confinement without affecting GAL1 transcription. Accordingly, transcriptional activation is necessary but not sufficient for the confinement of GAL genes at the nuclear periphery. The observed real-time dynamic mooring of active GAL genes to the inner side of the nuclear pore complex is in accordance with the 'gene gating' hypothesis.

Nuclear export of ribosomes requires a subset of nucleoporins and the Ran system, but specific transport factors have not been identified. Using a large subunit reporter (Rpl25p-eGFP), we have isolated several temperature-sensitive ribosomal export (rix) mutants. One of these corresponds to the ribosomal protein Rpl10p, which interacts directly with Nmd3p, a conserved and essential protein associated with 60S subunits. We find that thermosensitive nmd3 mutants are impaired in large subunit export. Strikingly, Nmd3p shuttles between the nucleus and cytoplasm and is exported by the nuclear export receptor Xpo1p. Moreover, we show that export of 60S subunits is Xpo1p dependent. We conclude that nuclear export of 60S subunits requires the nuclear export sequence-containing nonribosomal protein Nmd3p, which directly binds to the large subunit protein Rpl10p.Most steps in ribosome synthesis take place in the nucleolus, a specialized subnuclear region. This process starts with the synthesis of two pre-rRNA transcripts, 35S and pre-5S rRNA, which are processed and base modified to yield the mature 25S/28S, 18S, 5.8S, and 5S rRNAs, respectively (18). During these processes about 80 ribosomal proteins assemble onto the rRNAs to yield preribosomal particles, which are exported into the cytoplasm (41). In contrast to pre-rRNA processing and modification, very little is known about the assembly pathway for eukaryotic ribosomal subunits or the features that make them competent for nuclear exit (for recent reviews, see references 18 and 40).The transport of macromolecules through the nuclear pores is thought to involve facilitated diffusion of soluble transport factors over the repeat sequences of the nuclear pore proteins (nucleoporins) that form and line the nuclear pore complex. Directionality of transport is provided by the small GTPase Ran, due to the presence of a step RanGTP/RanGDP gradient across the nuclear membrane (for a review, see reference 23). RanGTP binds with high affinity to nuclear import and export receptors (importins and exportins, respectively) of the karyopherin ␤ superfamily (10). For nuclear exit, export cargoes, which harbor nuclear export sequences (NESs) (e.g., leucinerich NESs), form an intranuclear complex with the NES receptor Xpo1p/Crm1 in the presence of RanGTP (8,33). This trimeric complex is then exported from the nucleus into the cytoplasm.Saccharomyces cerevisiae has been a useful system for the analysis of the nuclear pore complex as well as transport factors (6). We have reported an in vivo assay for ribosomal export in yeast that uses a fusion between green fluorescent protein (GFP) and ribosomal protein Rpl25p (15). Rpl25p is imported into the nucleus and assembles with ribosomes by direct binding to the rRNA inside the nucleolus (39). Passage of both the free Rpl25p-GFP and the preribosomal particles through the nucleoplasm appears to be rapid in wild-type cells, and GFP-labeled ribosomes were detected by fluorescence microscopy in the cytoplasm. Mutations causing defects in subunit e...

The molecular mechanism underlying the retention of intron-containing mRNAs in the nucleus is not understood. Here, we show that retention of intron-containing mRNAs in yeast is mediated by perinuclearly located Mlp1. Deletion of MLP1 impairs retention while having no effect on mRNA splicing. The Mlp1-dependent leakage of intron-containing RNAs is increased in presence of ts-prp18 delta, a splicing mutant. When overall pre-mRNA levels are increased by deletion of RRP6, a nuclear exosome component, MLP1 deletion augments leakage of only the intron-containing portion of mRNAs. Our data suggest, moreover, that Mlp1-dependent retention is mediated via the 5' splice site. Intriguingly, we found Mlp-proteins to be present only on sections of the NE adjacent to chromatin. We propose that at this confined site the perinuclear Mlp1 implements a quality control step prior to export, physically retaining faulty pre-mRNAs.

A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export. Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm. Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins. These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export. Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified. Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export.