An endoribonuclease functionally linked to perinuclear mRNP quality control associates with the nuclear pore complexes

Abstract

Nuclear mRNA export is a crucial step in eukaryotic gene expression, which is in yeast coupled to cotranscriptional messenger ribonucleoprotein particle (mRNP) assembly and surveillance. Several surveillance systems that monitor nuclear mRNP biogenesis and export have been described, but the mechanism by which the improper mRNPs are recognized and eliminated remains poorly understood. Here we report that the conserved PIN domain protein Swt1 is an RNA endonuclease that participates in quality control of nuclear mRNPs and can associate with the nuclear pore complex (NPC). Swt1 showed endoribonuclease activity in vitro that was inhibited by a point mutation in the predicted catalytic site. Swt1 lacked clear sequence specificity but showed a strong preference for single-stranded regions. Genetic interactions were found between Swt1 and the THO/TREX and TREX-2 complexes, and with components of the perinuclear mRNP surveillance system, Mlp1, Nup60, and Esc1. Inhibition of the nuclease activity of Swt1 increased the levels and cytoplasmic leakage of unspliced aberrant pre-mRNA, and induced robust nuclear poly(A)(+) RNA accumulation in mlp1Delta and esc1Delta strains. Overexpression of Swt1 also caused strong nuclear poly(A)(+) RNA accumulation. Swt1 is normally distributed throughout the nucleus and cytoplasm but becomes concentrated at nuclear pore complexes (NPCs) in the nup133Delta mutant, which causes NPC clustering and defects in mRNP export. The data suggest that Swt1 endoribonuclease might be transiently recruited to NPCs to initiate the degradation of defective pre-mRNPs or mRNPs trapped at nuclear periphery in order to avoid their cytoplasmic export and translation.

Figure 1. Swt1 Has Endonucleolytic Activity on Single-Stranded RNA In Vitro

(A) Purified wild-type Swt1 and mutant Swt1-D135N proteins used for in vitro nuclease activity measurements. The indicated FLAG-tagged Swt1 proteins were affinity-purified from yeast and analyzed by SDS-PAGE and Coomassie staining. M, molecular weight marker. (B) Wild-type Swt1 or mutant Swt1-D135N protein (0.5 μg each) were incubated with 5′-[³²P]-end–labeled (left panel) or 3′-[³²P]-end–labeled (right panel) oligo(A)₃₀ RNA for 0, 60, and 120 min at 30 °C. The reaction products were separated on a denaturing 12% polyacrylamide/8 M urea gel and visualized by autoradiography. RNA probes incubated without protein served as negative controls, indicated by the negative sign (−). The asterisk (*) denotes a low activity of a contaminating yeast exonuclease. (C) Time course of Swt1 RNase activity on 5′-[³²P]-end–labeled (left panel) or 3′-[³²P]-end–labeled (right panel) oligo(A)₃₀ RNA. The assay was performed as described in (B), and aliquots were taken at the indicated time points. (D) Wild-type Swt1 or mutant Swt1-D135N were incubated with 5′-[³²P]-end–labeled 52-nt RNA with a stable terminal stem for 0, 30, and 60 min at 30 °C, and the reaction was analyzed as outlined in (B). OH, a partial alkaline hydrolysis of the RNA probe used as a molecular weight ladder. The positions of the stem residues are indicated in blue on the right side of the gel and on the RNA secondary structure calculated by MFOLD.

Figure 2. SWT1 Is Involved in Mlp1-Nup60-Assisted Nuclear mRNP Quality Control

(A) Disruption of SWT1 induces or enhances the nuclear accumulation of poly(A)⁺ and SSA1 RNA in mlp1Δ, esc1Δ, or nup60Δ strain, respectively. The indicated single- and double-mutant strains were grown at 30 °C (nup60Δ) or shifted to 37 °C for 90 min (mlp1Δ and esc1Δ), and the localization of poly(A)⁺ RNA and SSA1 RNA was analyzed by in situ hybridization using Cy3-oligo(dT) and SSA1-specific probes, respectively. DNA was stained with DAPI. The percentage of the cells showing a nuclear accumulation of poly(A)⁺ RNA or SSA1 mRNA, respectively, was calculated for n > 150 cells and indicated below each panel (mlp1Δ and esc1Δ strains). In the case of the nup60Δ strain, the percentage of cells with a significantly enhanced nuclear poly(A)⁺ signal (above 1.5-fold of the average intensity of the nup60Δ nuclear signal) is shown. (B) Deletion of SWT1 induces a leakage of reporter pre-mRNAs to the cytoplasm and synergistically enhances the pre-mRNA leakage of mlp1Δ and nup60Δ cells. The leakage of unspliced lacZ pre-mRNA reporters that give rise to β-galactosidase activity was analyzed as described in Materials and Methods. The ratios between β-galactosidase activities derived from the reporter pre-mRNA and intronless control mRNA were calculated and shown relatively to the ratio of respective wild-type strain. The results of four independent experiments are shown. Error bars indicate standard deviations.

Figure 3. The Catalytically Inactive swt1-D135N Mutant Resembles the swt1 Null Mutation In Vivo

(A) The swt1-D135N mutant does not complement the synthetically lethal/enhanced phenotypes of the indicated swt1Δ double-mutant strains. Strains were transformed with wild-type SWT1 or mutant swt1-D135N, and transformants were spotted in 10-fold serial dilutions on the indicated plates. The strains were incubated for 5 d at 30 °C (tho2Δ swt1Δ) or for 3 d at 36 °C (thp2Δ swt1Δ and nup60Δ swt1Δ). (B) The expression levels of wild-type and mutant Swt1 proteins are similar. Whole-cell lysates prepared from a swt1Δ strain or a swt1Δ strain expressing wild-type Swt1 or Swt1-D135N were analyzed by SDS-PAGE and western blotting using polyclonal anti-Swt1 antibodies. The detection of Arc1 protein by an anti-Arc1 polyclonal antibody was used as a loading control. (C) The Swt1-D135N mutant cannot rescue the poly(A)⁺ export defect of the mlp1Δ swt1Δ strain. mlp1Δ swt1Δ cells expressing wild-type Swt1 or Swt1-D135N were analyzed for poly(A)⁺ RNA localization as in Figure 2A or stained with DAPI to detect DNA. (D) Swt1-D135N-expressing cells show a nuclear pre-mRNA leakage phenotype. The swt1Δ and mlp1Δ swt1Δ strains were transformed with the indicated plasmids and assayed for the leakage of the lacZ pre-mRNA reporter (mutBP) as described in Figure 2B. The results of four independent experiments are shown. Error bars indicate standard deviations. (E) Swt1-D135N cells accumulate elevated levels of mutated lacZ pre-mRNA. Total RNA was isolated from the swt1-D135N and isogenic wild-type strain expressing the intron-containing lacZ reporter transcript (norm., for normal) or the intron-containing lacZ transcript mutated in the branch-point splicing site (mut., for mutated). The lacZ levels were quantified by real-time RT-PCR using the GAL1 mRNA as a reference transcript. The results of four independent experiments are shown. Error bars indicate standard errors of the mean. (F) Overexpression of wild-type Swt1, but not overexpression of the catalytically inactive Swt1-D135N mutant, is toxic to the cells. Swt1Δ cells containing GAL1::SWT1, GAL1::swt1-D135N, or empty plasmid were spotted in 10-fold serial dilutions on plates containing either galactose or glucose and incubated for 3 d at 30 °C. Only the last two dilutions of glucose plate are shown. (G) Overexpression of wild-type Swt1, but not overexpression of the catalytically inactive Swt1-D135N mutant, induces strong nuclear accumulation of poly(A)⁺ RNA. The same strains as used in (F) were grown in raffinose-containing medium to early-log phase and then induced by 2% galactose for 5 h. The localization of poly(A)⁺ RNA was analyzed by in situ hybridization using a Cy3-oligo(dT) probe. DNA was stained with DAPI. The percentage of the cells showing an accumulation of nuclear poly(A)⁺ signal is shown (n > 150 cells).

Figure 4. Swt1 Associates with Nuclear Pore Complexes in the Nucleoporin Mutant nup133Δ

(A) 4xGFP-Swt1 (green) or (B) 4xGFP-Swt1ΔPIN (green) were coexpressed with Nup120-mCherry (a marker for NPCs; red) in the indicated strains (swt1Δ, nup133Δ swt1Δ, nup133Δ swt1Δ + NUP133 [expressed from a centromeric plasmid], mlp1Δ swt1Δ, and nup60Δ swt1Δ) and analyzed by fluorescence microscopy. Cells were also viewed by Nomarski optics.