Pub1 binds to proteins involved in RNA metabolism. (A) Yeast two-hybrid strain L40 (Clontech) containing a LexA-Pub1 plasmid (pSV424) was transformed with positive clones from the two-hybrid screen (GAR1, AIR1, NAB2, and HHT1) or with vector pACT (negative control). Transformants and positive control SVL129 cells (64) were grown on control synthetic complete (SC) plates or SC plates lacking histidine (−His) and also assayed for β-galactosidase activity (X-gal). Positive interactions are indicated by growth on plates lacking His and blue color on X-gal plates (darker grey in the right column). (B) Immunoblot analysis of unbound (UB) and bound (B) fractions from the copurification assay using cells expressing Nab2-TAP (lanes 1 to 4) or a control protein, Sec27-TAP (lanes 5 to 8). Proteins were purified with IgG-Sepharose in the presence or absence of 10 μg RNase A, and fractions were probed with an anti-Pub1 antibody (top) or peroxidase antiperoxidase antibody complex (Sigma) (bottom).

Deletion of PUB1 does not cause poly(A) RNA accumulation within the nucleus. PUB1 deletion cells (Δpub1) were grown to log phase and subjected to FISH as described in Materials and Methods. As a control, we also analyzed NAB2 deletion cells (SVL544) expressing either wild-type (WT) NAB2 (pSV572) or the nab2-1 allele (pSV575). Poly(A) RNA was detected using an oligo(dT) probe. The nucleus is indicated by DAPI staining of chromatin, and corresponding differential interference contrast images are shown.

Amino acid substitutions within the Nab2 zinc finger domain abolish the interaction with Pub1 and decrease RPS16B mRNA stability. (A) Schematic of amino acid changes within the Nab2 CCCH domain. The specific amino acid residues changed in each Nab2 variant are detailed in Table S1 in the supplemental material. (B) Amino acid substitutions in the Nab2 CCCH domain decrease binding to Pub1. Two-hybrid SVL316 (EGY48; Invitrogen) cells expressing a transcription activation domain-Pub1 fusion (B42-Pub1; pSV545) were transformed with vector (pEG202) (-) or plasmids expressing wild-type (+) or the indicated variant CCCH Nab2 domains. Cells were grown in liquid medium containing galactose, and quantification of lacZ reporter activity, as a measure of the Pub1 interaction with the CCCH domain of Nab2, was determined by an in vitro β-galactosidase assay. The bar graph displays β-galactosidase activity relative to the wild-type CCCH domain, which was set to 1.0. Standard deviations in the data are indicated. (C) Nab2-67 shows a greatly decreased interaction with Pub1. GST (lanes 1 and 2), GST-Nab2 (lanes 3 and 4), or GST-Nab2-67 (lanes 5 and 6) was purified from E. coli and incubated with recombinant His₆-Pub1. The unbound (UB) and bound (B) fractions were analyzed by immunoblotting with anti-Pub1 and anti-GST antibodies. The bottom panel corresponds to the unbound and bound bands from different areas in the blot due to differences in protein sizes (26 kDa for GST alone and 95 kDa for GST-Nab2). (D) The nab2-67 mutant allele is functional. A plasmid shuffle technique was used to assess the ability of the nab2-67 mutant to replace the essential function of NAB2. NAB2 deletion cells (SVL544) maintained by a URA3 NAB2 plasmid (pSV877) were transformed with test plasmids carrying vector alone (pSV59), wild-type NAB2 (pSV572), or nab2-67 (pSV876). Samples were serially diluted and spotted onto a control plate (SC medium lacking Ura), where the wild-type NAB2 plasmid is retained, or selective medium (5-fluoroorotic acid [5-FOA]), where the wild-type NAB2 plasmid is lost and the only cellular copy of the essential NAB2 gene is provided by the test plasmid. Plates were incubated for 3 days at 30°C. Vector alone and wild-type NAB2 served as the negative and positive growth controls. (E) The stability of the RPS16B transcript is decreased in nab2-67 cells compared to wild-type (WT) cells. The half-lives of the RPS16B and GCN4 transcripts were determined using qRT-PCR as described in Materials and Methods.

Pub1 binds directly to the zinc finger domain of Nab2. (A) Schematic of Nab2 mutant proteins where the full length (amino acids 1 to 524) is shown at the top and where the four domains within the wild-type protein are indicated by the shading. The amino acids contained within the variant proteins are indicated. (B) Two-hybrid analysis to define the Pub1-interacting domain of Nab2. Two-hybrid SVL316 (EGY48; Invitrogen) cells expressing a transcription activation domain-Pub1 fusion (B42-Pub1; pSV545) transformed with vector (pEG202) (negative control) or plasmids expressing each Nab2 variant were grown on X-gal plates containing galactose to induce the expression of the Pub1 fusion protein. Positive interactions are indicated by the darker grey. (C) GST (lanes 1 and 2), GST-Nab2 (lanes 3 and 4), GST-CCCH (lanes 5 and 6), and GST-Nab2 ΔCCCH (lanes 7 and 8) were purified from E. coli and incubated with recombinant His₆-Pub1. The unbound (UB) and bound (B) fractions were analyzed by immunoblotting with antibodies against Pub1 and GST, as described in Materials and Methods. The bottom panel, which shows each of the GST fusion proteins, displays the unbound and bound bands from different areas of the blot due to differences in protein sizes (26 kDa for GST alone, 95 kDa for GST-Nab2, 59 kDa for GST-CCCH, and 62 kDa for GST-ΔCCCH).

Nab2 modulates mRNA stability of an ARE-like sequence-containing transcript. (A) Schematic of the ARE-like sequence-containing RPS16 and STE-containing GCN4 transcripts. (B) The stability of the RPS16B transcript is decreased in nab2-1 cells. Wild-type (WT), Δpub1, nab2-1, Δpub1 nab2-1, and rat7-1 cells harboring a temperature-sensitive allele of RNA polymerase II (rpb1-1) (43) were grown to mid-log phase in YPD medium, and total mRNA was isolated at specific times following the inhibition of transcription by shifting the cells to the nonpermissive temperature. cDNA was prepared from each sample and subjected to qRT-PCR to analyze the RPS16B, GCN4, and PGK1 (stable control) transcripts. Transcript half-lives were calculated by exponential fit, and a P value of <0.05 was considered to be significant. Standard deviations are indicated. (C) Northern blot analysis of decay of the RPS16B transcript. Total mRNA samples isolated after shifting wild-type, Δpub1, and nab2-1 cells harboring the rpb1-1 allele to the nonpermissive temperature were probed for RPS16B and PGK1 transcripts at the times indicated.

Stabilization of RPS16B by Nab2 is dependent on the presence of the ARE-like sequence. (A) Schematic of integration at the RPS16B locus demonstrating how the integration of RPS16B-ΔARE was accomplished. The location of the primer pairs used to simultaneously amplify both endogenous RPS16B and RPS16B-ΔARE by qRT-PCR is also indicated. The F primer, which is used to amplify both endogenous RPS16B and the integrated RPS16B-ΔARE allele, hybridizes within the ORF region of both RPS16B sequences. The R primer, which specifically amplifies endogenous RPS16B, hybridizes with the ARE element. The R′ primer, which specifically amplifies RPS16B-ΔARE, hybridizes to the junction created by the deletion of the ARE element. (B) The half-lives of both the RPS16B and RPS16B-ΔARE transcripts were simultaneously determined in wild-type (WT), Δpub1, nab2-1, and nab2-67 cells harboring the rpb1-1 allele. Cells were grown to mid-log phase in YPD medium and shifted to the nonpermissive temperature for 0, 2, 5, 10, 15, and 20 min. Total mRNA was isolated from each sample, reverse transcribed to cDNA, and subjected to qRT-PCR to determine RPS16B, RPS16B-ΔARE, and PGK1 (control) transcript half-lives. Standard deviations in the data are indicated.