Crp79p, like Mex67p, is an auxiliary mRNA export factor in Schizosaccharomyces pombe

Abstract

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)(+) RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.

Figure 1

Suppression of the mRNA export defect in SL27 (rae1–167 nup184–1/pRXE81X-rae1) cells. Poly(A)⁺ RNA localization in SL27 cells and in cells with the Δcrp79 mutation. (A) Poly(A)⁺ RNA was detected in Δcrp79 cells grown in EMM medium. The SL27 cells carrying an empty vector or a plasmid (p27–9) expressing Crp79p were grown in EMM to midlog phase expressing rae1 (absence, −B1) or without expression of rae1 (18 h in presence of 10 μM B1). Coincident DAPI staining is shown at bottom. (B) aa sequence and structural organization of Crp79p of S. pombe. The sequence of Crp79p has been deposited in the GenBank data base with accession No. AF432874. Shaded boxes indicate the three RRM domains. The secondary structure predictions of α-helix and β-sheets are indicated and underlined (Burd and Dreyfuss, 1994a). The dark underline indicates the position of the leucine-rich NES.

Figure 2

Growth and mRNA export of SL27 cells. (A) Complementation of synthetic lethality of rae1–167 nup184–1/pREP81X-rae1 (SL27) by Crp79p-GFP and its deletions. A schematic diagram of the regions surrounding the deletions along with deletion endpoints is shown (for details of plasmid construction, see MATERIALS AND METHODS). Growth of SL27 cells was monitored for 4 d with (−B1) and without (+B1) expression of rae1. Ability and inability to restore growth and mRNA export to SL27 cells under conditions of synthetic lethality is indicated and summarized by (+) and (−). The GFP loc. indicates cellular localization of relevant fusion protein; C, cytoplasm; N, nucleus; N/A, not applicable. (B) Determination of steady-state protein levels of Crp79p-GFP and deletion derivatives in SL27 cells grown when expression of rae1 was repressed (in presence of B1). Top, Western blot analysis using a monoclonal anti-GFP antibody. Bottom, Control for protein loading by Western blot for a protein that reacts with NPC-specific mAb414 mAb. (C) Poly(A)⁺ RNA was detected in SL27 cells carrying pAB11, pAB12, pAB115, and pAG157 (see Figure 2A for deletion endpoints) grown in EMM to midlog phase in absence (−B1) or 18 h in presence of thiamine (10 μM). Coincident DAPI staining is shown at bottom.

Figure 3

UV cross-linking and localization of Crp79p-GFP. (A) Crp79-GFP and N-terminal GST-fusions of RRM1, RRM1 F74V, RRM2, and RRM3 were expressed from plasmids under the control of the genomic crp79 promoter or a heterologous (rae1) promoter, respectively. UV cross-linked (+UV) or uncross-linked (−UV) samples were prepared. The protein cross-linked to poly(A)⁺ RNA were detected by Western blot analysis using polyclonal anti-GST antibody. (B) Cells in which Crp79-GFP was integrated at its genomic locus were tested for GFP signal. For cellular localization of Crp79-GFP (pAB3) or a deletion carrying RRM1 and RRM2 fused to GFP (pAG157) in SL27 cells, transformants were grown with or without rae1 expression (−B1 or 18 h in presence of B1), and the proteins were visualized by GFP fluorescence. Coincident DAPI staining is shown at bottom.

Figure 4

Identification of nuclear export activities in Crp79p using a heterologous assay for protein export. (A) A schematic map of different deletions used in the nuclear export assay in B is indicated. (B) The regions of Crp79p as indicated were expressed as hormone-inducible Gr-GFP chimera proteins in HeLa cells. Import: treatment with 2 μM corticosteroid for 1 h at 37°C. Export: cells were washed three times with phosphate-buffered saline to remove hormone and incubated for an additional hour at 37°C in the presence of cycloheximide (10 μg/ml). spMex67(NES)-Gr-GFP was used as a positive control for the assay (Yoon et al., 2000).

Figure 5

Nuclear export activity–dependent suppression of growth and mRNA export defects of SL27 cells. (A) Schematic diagram of various GFP fusions. The coordinates for the spMex67 are aa 439–505 (Yoon et al., 2000). The Rev-NES sequence is 23 aa long, containing LPPLERLTL (wild-type) or LPPAERATL (mutant) peptide sequence (Love et al., 1998). Growth of rae1–167 nup184–1 cells was monitored for 4 d, expressing different GFP fusion constructs with or without rae1 expression (−B1 and +B1, respectively). + indicates ability to complement, and − indicates inability to complement under synthetic lethal conditions. The GFP loc. indicates cellular localization of relevant Crp79p-GFP fusion; C, cytoplasm; N, nucleus; and N/A, not applicable. (B) Steady-state levels of GFP fusion proteins used in A following a procedure similar to that used in Figure 2B. (C) Poly(A)⁺ RNA localization of various GFP fusions in A as indicated in SL27 cells after 18 h of growth in presence of B1 (10 μM) are shown. (D) Localization of the GFP fusions in A as indicated in the absence (−B1) or 18 h after addition of B1 to the media. Coincident DAPI staining is shown at bottom.

Figure 6

Shuttling of Crp79p-GFP and Ran-dependent import. (A) Shuttling of Crp79p-GFP fusion in rae1–167 cells. The nuclear accumulation of Crp79p-GFP in cells with rae1–167 mutation at 27°C (a) and after shift to 36°C for 30′, 60′, and 90′ (b–d) are indicated. Incubation of cells at 36°C for 90 min followed by additional incubation for 150 min at a permissive temperature of 25°C is shown in e. Localization of SV40-NLS-lacZ-GFP was used as a control; cells were incubated at 36°C followed by incubation at 25°C for 150 min (f). Coincident DAPI staining for all samples are shown at bottom (g–i). (B) Localization of Crp79p-GFP and SV40-NLS-lacZ-GFP in rae1–167 pim1-d1 strain after simultaneous inactivation of both proteins for 90 min at 36°C are shown. Coincident DAPI staining is shown below.

Figure 7

Schematic summarizes the functional domains of Crp79p. A weak leucine-rich NES present in RRM2 is indicated, and the coordinates of the minimal and optimal nuclear export activity (NES) as identified in HeLa cells are indicated.