A Yeast RNA-Binding Protein Shuttles between the Nucleus and the Cytoplasm

Abstract

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.

Saccharomyces cerevisiae Ntg1p and Ntg2p: Broad Specificity N-Glycosylases for the Repair of Oxidative DNA Damage in the Nucleus and Mitochondria

“…Polyclonal antibodies against GFP and Npl3p were generous gifts from Drs. J. Kahana and P. Silver (13,14). E. coli endonuclease III and endonuclease VIII were prepared and purified as previously described (15,16).…”

“…Indirect immunofluorescence microscopy was employed to confirm the nuclear localization of Ntg2p. For these experiments cells were grown in SD-URA at 30 °C to a concentration of 1 × 10 7 cells/mL and were prepared for indirect immunofluorescence microscopy as described previously (13). Briefly, cells were fixed in 3.7% formaldehyde for 30 min, washed in 0.1 M potassium phosphate buffer, pH 6.5, and resuspended in P solution (0.1 M potassium phosphate buffer, pH 6.5, 1.2 M sorbitol).…”

“…To confirm that Ntg2p is a strictly nuclear protein, immunofluorescence using an antibody to a nuclear protein, Npl3p, was utilized (13). As seen in Figure 2B, Ntg2p-GFP localizes to the nucleus (panels A and F).…”

Reporters of Gene Expression: Autofluorescent Proteins

“…Since making its début as a reporter of gene expression in bacteria and worms (Chalfie et al, ), GFP and its variants have been expressed in a remarkably diverse range of organisms. The creation of transgenic GFP models in plants (Baulcombe et al, ), yeast (Flach et al, ), mosquito (Higgs et al, ), Drosophila (Wang and Hazelrigg, ; Barthmaier and Fyrberg, ), zebrafish (Amsterdam et al, ), Dictyostelium discoideum (Maniak et al, ), Xenopus (Tannahill et al, ), and mouse (Ikawa et al, ) attests to their utility. Furthermore, expression of red‐shifted GFP in retroviruses used in hemopoietic reconstitution assays (Persons et al, ; Rasko et al, ) and many other examples has enabled tracking of mature cells of different phenotypes which arose from common precursors.…”

pH-Dependent Fluorescence of a Heterologously Expressed Aequorea Green Fluorescent Protein Mutant: In Situ Spectral Characteristics and Applicability to Intracellular pH Estimation,

“…This has led to a rapid sucession of novel and useful molecular biology applications for both wild-type and mutant GFPs [for review, see (5)]. GFPs have been used as reporters of gene expression (2,(6)(7)(8), as markers of cell lineage (5), and as peptide fusion tags to monitor both intracellular peptide localization (9)(10)(11) and protein trafficking (12)(13)(14). Their small size [∼27 kDa (15)], nonperturbing nature, and remarkable stability under a variety of denaturing conditions (16)scoupled with their ability to retain their intrinsic fluorescence when fused to other peptidesshas made them an indispensable part of the molecular biology armamentarium.…”