No abstract

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit in S. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ϳ45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b-GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNAprocessing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b-GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b-GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b-GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120. INTRODUCTIONAlthough eukaryotic ribosomes function in the cytoplasm, the synthesis, processing, and assembly of the ribosomal subunits in Saccharomyces cerevisiae and higher eukaryotes occur in the nucleolus. The entire ribosome is composed of four rRNA species and ϳ75 ribosomal proteins (r-proteins) distributed between two subunits. The 18S, 5.8S, and 25S rRNAs are derived from a single 35S rRNA precursor that is synthesized by RNA polymerase I and then processed by a series of endonucleolytic and exonucleolytic cleavages (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999). The 5S rRNA is synthesized separately by RNA polymerase III and associates with the 60S preribosomal subunit early in assembly. The mature 40S ribosomal subunit contains the 18S rRNA and ϳ32 r-proteins, whereas the 60S subunit is composed of the 5S, 5.8S, and 25S rRNAs and ϳ45 r-proteins. Proper assembly of each ribosomal subunit requires the coordination of several events, including the synthesis and import of r-proteins, the synthesis and processing of rRNA, and the concomitant assembly of r-proteins into the preribosomal subunits. Although a pathway for 35S rRNA maturation has been well defined through both genetic and biochemical approaches (reviewed by Kressler et al., 1999;Venema and Tollervey, 1999), less is known about the association of r-proteins with the rRNA and the export of the assembled subunits out of the nucleus.All nucleocytoplasmic transport occurs ...

A number of RNA-binding proteins are associated with mRNAs in both the nucleus and the cytoplasm. One of these, Npl3p, is a heterogeneous nuclear ribonucleoprotein-like protein with some similarity to SR proteins and is essential for growth in the yeast S. cerevisiae. Temperature-sensitive alleles have defects in the export of mRNA out of the nucleus (1). In this report, we define a genetic relationship between NPL3 and the nonessential genes encoding the subunits of the cap-binding complex (CBP80 and CBP20). Deletion of CBP80 or CBP20 in combination with certain temperature-sensitive npl3 mutant alleles fail to grow and thus display a synthetic lethal relationship. Further evidence of an interaction between Npl3p and the cap-binding complex was revealed by co-immunoprecipitation experiments; Cbp80p and Cbp20p specifically co-precipitate with Npl3p. However, the interaction of Npl3p with Cbp80p depends on both the presence of Cbp20p and RNA. In addition, we show that Cbp80p is capable of shuttling between the nucleus and the cytoplasm in a manner dependent on the ongoing synthesis of RNA. Taken together, these data support a model whereby mRNAs are co-transcriptionally packaged by proteins including Npl3p and capbinding complex for export out of the nucleus.While in the nucleus, mRNA precursors, referred to as pre-mRNAs or heterogeneous nuclear RNAs undergo a series of processing events before entering the cytoplasm. These maturation events include co-transcriptional capping at the 5Ј-end, splicing, and cleavage and polyadenylation at the 3Ј-end. The proper execution of these steps affects the export of mRNA (reviewed in Refs. 2-4). Thus, the process of mRNA export commences long before the RNA actually reaches the nuclear membrane.