Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae.

Abstract

Abstract. To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nablp, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A) + RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nablp is identical to the Npl3p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and prerRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TP//mRNAs, a concomitant accumulation of unspliced ACTI pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing. HETEROGENEOUS nuclear RNAs (hnRNAs) t are the products of RNA polymerase II transcription, and include polyadenylated and nonpolyadenylated premRNAs and mRNAs as well as several uncharacterized RNAs (Dreyfuss et al., 1993). An array of nuclear factors bind to hnRNAs during transcription, including heterogeneous nuclear ribonucleoproteins (hnRNPs) and small nuclear RNP (snRNP) particles (Amero et al., 1992;Matunis et al., 1993). Numerous studies have established the roles of snRNP particles in a variety of nuclear processes such as pre-mRNA splicing (Green, 1991;Guthrie, 1991; Ruby and Abelson, 1991;Baserga and Steitz, 1993). In contrast, the functions of hnRNPs have remained unclear. HnRNPs are nuclear RNA-binding proteins whose primary and stable RNA-binding site is hnRNA. Current ideas about the functional roles of hnRNPs in pre-mRNA processing have Address all correspondence to M.

The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA

“…As expected for an integral subunit of eIF3, HA-Tif34p was found exclusively in the cytoplasm (Fig. 5B, k), whereas Nab1p showed diffuse nuclear staining characteristic of a nucleoplasmic protein (panel e) (Wilson et al 1994). Both HA-Gcd10p and HA-Gcd14p showed prominent nuclear localization with staining indicative of nucleoplasmic factors (Fig.…”

“…The affinity-purified 12CA5 monoclonal antibody against the HA epitope (at 20 µg/ml; Boehringer Mannheim) was used to probe strains expressing HA-tagged proteins and the isogenic control strains lacking tagged proteins (a,c,g,i,k). Monoclonal antibody 1E4 (at 1:750 dilution; Wilson et al 1994) was used to detect Nab1p in strain YJA142 (e). Detection of the primary antibodies was accomplished using a fluorescein isothiocyanate (FITC)-conjugated secondary antibody (a,c,e,g,i,k) and the DNA distribution was visualized by DAPI (b,d,f,h,j,l).…”

Arginine methylation facilitates the nuclear export of hnRNP proteins

“…Both Npl3p and Hrp1p bind poly(A) + RNA (Wilson et al 1994;Kessler et al 1997). Hrb1p was also examined for its ability to bind to poly(A) + RNA by irradiation of cells with ultraviolet light to crosslink RNA to proteins.…”

Importin/karyopherin protein family members required for mRNA export from the nucleus

“…3A Lower). We also examined the localization of the H2B-NLS fused to ␤-galactosidase (35), Nop1p (38), Nab2p (39), Nab3p (40), and Hrp1p (41) in pse1-1 ⌬kap123 cells and found them all to remain nuclear at all temperatures (data not shown). To account for the possibility that these endogenous proteins are not synthesized at sufficiently high levels at the nonpermissive temperature because of the mRNA export block, we also analyzed the re-import of a SV40 NLS-containing GFP fusion protein (NLS-GFP; ref.…”