Yra1p, a conserved nuclear RNA-binding protein, interacts directly with Mex67p and is required for mRNA export

Abstract

Mex67p and Mtr2p constitute an essential mRNA export complex that interacts with poly(A)+ RNA and nuclear pore proteins. We have identified Yra1p, an intranuclear protein with in vitro RNA-RNA annealing activity, which directly binds to Mex67p. The complex between Yra1p and Mex67p was reconstituted in vitro and shown by UV-crosslinking to bind directly to RNA. Mutants of YRA1 are impaired in nuclear poly(A)+ RNA export at restrictive growth conditions. ALY, the mouse homologue of Yra1p and a transcriptional coactivator, can bind in vitro to yeast and human Mex67p and partly complements the otherwise non-viable yra1 null mutant. Thus, Yra1p is the first RNA-binding protein characterized, which bridges the shuttling Mex67p/Mtr2p exporter to intranuclear mRNA transport cargoes.

Fig. 1. A novel mex67-6 ts allele with mutations in the N+LRR domain is defective in nuclear mRNA export. (A) Domain organization of Mex67p and amino acid mutations within the mex67-5 (Segref et al., 1997) and mex67-6 allele. The Mex67p protein consists of an N–terminal (N), LRR, middle (M) and C–terminal (C) domain with the indicated amino acid boundaries. (B) Growth of the mex67-6 ts mutant. Equivalent numbers of cells were diluted in 10^–1 steps, spotted onto YPD plates, and grown for 3 days at the temperatures indicated. (C) Nuclear accumulation of polyadenylated RNA in mex67-6 cells. Subcellular localization of poly(A)⁺ RNA was analyzed by in situ hybridization. MEX67 and mex67-6 cells were either grown at 23°C or shifted for 1 h to 37°C. Nuclear DNA was stained with 4′-6-diamidine-2-phenylindole (DAPI).

Fig. 2. Identification of YRA1, which functionally interacts with the N+LRR domain of MEX67. (A) The synthetically lethal mutant sl59 is complemented by YRA1. Red/white colony sectoring is restored in the mex67-6 synthetically lethal strain sl59 by transformation with plasmid-borne MEX67 (+pMEX67) or YRA1 (+pYRA1), but not with an empty plasmid (+pUN100). sl59 is also complemented by homologous integration of the cloned YRA1 gene (+YRA1::LEU2). (B) The sl59 mutant is also complemented by MTR2 and mex67-5. sl59 carrying plasmids pURA3-MEX67 and pTRP1-mex67-6 is unable to grow in the presence of 5–FOA, unless complemented either by transformation with wild-type MEX67, YRA1, or by integration of wild-type YRA1 into the yra1 locus of sl59. sl59 was also transformed with empty pUN100, pRS315-MTR2, pRS315-mex67-6 or pUN100-mex67-5. Transformants were streaked onto 5–FOA containing plates and grown for 3 days at 30°C. (C) Synthetic lethal interaction between mex67-6 and yra1-1 alleles. The yra1::HIS3/mex67::HIS3 shuffle strain (Table I) was transformed with plasmids giving the gene combinations mex67-6/yra1-1, mex67-6/YRA1 and MEX67/yra1-1. A synthetic lethal relationship was tested by streaking transformants on 5–FOA–containing plates.

Fig. 3. Yra1p is an intranuclear protein. (A) Fluorescence microscopy of yra1::HIS3 cells expressing GFP–Yra1p. Fluorescence microscopy and Nomarski pictures were merged. (B) Fluorescence microscopy of GFP–Yra1p and GFP–Mex67p, respectively, in nup133^– cells. (C) GFP–Yra1p does not shuttle between the nucleus and the cytoplasm. The reporter constructs pGAL1::GFP–Npl3, pGAL::GFP–Yra1p, and NLS–GFP were expressed in the nup49-313 strain. Expression was stopped by shifting the cells to glucose-containing medium (not in the case of NLS–GFP, which has a constitutive promoter). Strains were further incubated at 23°C (permissive temperature) or shifted for 5 h to 37°C (restrictive temperature), before the intracellular location of GFP fusion proteins was analyzed by fluorescence microscopy. In all cases, the same exposure time was used.

Fig. 4. YRA1 is essential for nuclear poly(A)⁺ RNA export. (A) Growth curve of the GAL1::GFP–YRA1 strain in galactose (YPGal) and glucose-containing medium (YPD) by measuring optical density at 600 nm. (B) Repression of GFP–Yra1p expression as determined by Western blot analysis using anti-GFP antibodies. The same amount of whole-cell extract derived from strain GAL1::GFP–YRA1 was analyzed after 0, 5 and 12 h in glucose-containing medium. (C) Subcellular localization of poly(A)⁺ RNA in GAL1::GFP–YRA1 cells was analyzed by in situ hybridization with a fluorescently labeled poly(dT) probe. The strain was grown in galactose-containing medium prior to transfer to glucose-containing medium for 5 and 12 h. Nuclear DNA was stained with DAPI. (D) Growth of the yra1-1 ts mutant. The same amount of yra1-1 and YRA1 cells, respectively, were diluted in 10^–1 steps, spotted onto YPD plates, and grown for 3 days at 30 or 37°C. (E) Nuclear accumulation of poly(A)⁺ RNA in yra1-1 cells, as determined by in situ hybridization. yra1-1 cells were either grown at 18°C or shifted for 1 h to 37°C in YPD medium. Nuclear DNA was stained with DAPI, and Nomarski pictures were taken. A schematic representation of the yra1-1 allele with its five mutations in the context of the Yra1p domain organization (see also Figure 7) is also shown.

Fig. 5. Yra1p associates with Mex67p both in vivo and in vitro. (A) Affinity purification of ProtA–Yra1p. The whole-cell lysate (7.5 μl) (lane 1), the unbound fraction (lane 2) and the eluate from the IgG–Sepharose beads (lane 3) were analyzed by SDS–PAGE followed by Coomassie Blue staining, or Western blotting using anti-ProtA antibodies. (B) Western blot analysis of purified Yra1–ProtA (lane 1) and a whole cell lysate from strain RS453 (lane 2) using anti-Mex67p and anti-Nup85p antibodies. (C) In vitro binding of Mex67p to GST-tagged Yra1p. Bead-immobilized GST–Yra1p or GST–Npl3p purified from S.pombe was incubated with E.coli whole-cell lysates containing Mex67p/Mtr2p or Mtr2p or buffer. Bound proteins were analyzed by SDS–PAGE, followed by Coomassie Blue staining or Western analysis using anti-Mtr2p antibodies (lower panel). The positions of GST–Yra1p and GST–Npl3p, Mex67p (filled triangle), and an E.coli contaminating band (asterisk) are indicated. Lane 1, protein standard; lane 2, E.coli lysate with Mex67p/His₆-Mtr2p complex; lane 3, E.coli lysate with His₆-Mtr2p; lane 4, GST–Yra1p incubated with Mex67p/His₆-Mtr2p lysate (–RNase); lane 5, GST–Yra1p incubated with Mex67p/His₆-Mtr2p lysate (+RNase); lane 6, GST–Yra1p incubated with His₆-Mtr2p lysate; lane 7, GST–Yra1p incubated with buffer; lane 8, GST–Npl3p incubated Mex67p/His₆-Mtr2p lysate; lane 9, GST–Npl3p incubated with buffer.

Fig. 6. Yra1p has RNA–RNA annealing activity and can be UV-crosslinked together with Mex67p to RNA. (A) RNA–RNA annealing assay. In vitro transcribed SSA1 sense (³²P-labeled) and anti-sense (non-labeled) RNAs were incubated with or without recombinant, purified Yra1p. RNA–RNA hybrid formation was analyzed by digestion of single-stranded RNA with RNase T₁. Double-stranded and thus protected RNA fragments were separated on a 10% polyacrylamide/8 M urea gel, before autoradiography was performed. Lane 1, ∼40 ng Yra1p with both ³²P-labeled and unlabeled RNA; lane 2, ∼20 ng Yra1p with both ³²P-labeled and unlabeled RNA; lane 3, ∼40 ng Yra1p with only ³²P-labeled RNA; lane 4, no Yra1p, but both ³²P-labeled and unlabeled RNA; lane 5, untreated sense ³²P–labeled RNA. (B) UV-crosslinking of RNA to Yra1p and Mex67p. ³²P-labeled SSA1 RNA (bp 818–1152) was incubated with GST, GST–Yra1p on beads, and GST–Yra1p bound to Mex67p/Mtr2p on beads. After UV-irradiation, the non-crosslinked RNA was digested with RNase A and RNase T₁, and proteins were analyzed by SDS–PAGE, followed by Coomassie Blue staining and autoradiography. Lane 1, GST; lane 2, GST–Yra1p bound to beads; lane 3, GST–Yra1p with bound recombinant Mex67p/Mtr2p complex on beads.

Fig. 7. Mouse ALY complements the yra1^– mutant. Upper panel: Schematic representation of the Yra1p/ALY domain organization, which includes a short N-terminal domain (N), a RGG box (only in ALY, but not Yra1p), and RNA-binding domain (RRM), a putative NLS and a C-terminal (C) domain; the different YRA1 homologues (including ALY) are aligned. Lower panel: restoration of growth of the yra1::HIS3 strain by expression of mouse ALY. The YRA1 shuffle strain (Table I) was transformed with 2μ plasmids pNOPA2A-ALY (ALY), pNOPA2A empty (empty plasmid), or ARS–CEN plasmid pNOPGFPA-YRA1 (YRA1). In the case of ALY, two individual transformants were first grown on selective SDC –ade plates, and then restreaked on 5-FOA. On this plate, only cells that have lost the pURA3-YRA1 plasmid, and thus are yra1::HIS3 can grow. When ALY is expressed, small colonies became visible after 3 days incubation at 30°C. Plates were incubated for 13 days, prior to being photographed.