Nab2, Yra1, and Mex67 form a trimeric complex. (A) Nab2 coimmunoprecipitates Mex67 and Yra1 in vivo in an RNA-independent manner. Yeast extracts from Nab2-ProtA or nontagged (No tag) cells and expressing HA-tagged Cbp20 were treated (+) or not (−) with RNases and purified with IgG-Sepharose beads. Input and eluted fractions (IP) were analyzed by Western blotting with anti-Mex67, anti-Yra1, anti-HA, anti-Rap1, anti-Actin, and anti-ProtA antibodies. (B) Yra1 promotes binding of Nab2 and Mex67. GST-Mex67 on beads was incubated with purified His₆-Nab2 (lane 1), His₆-Nab2 plus equimolar (lane 2) or 10× molar (lane 3) amounts of purified His₆-Yra1, BSA (lane 4), purified His₆-Yra1 alone (lane 5), or buffer alone (lane 6). His₆-Nab2 and His₆-Yra1 input are in lanes 7 and 8, respectively. Western blot (top panel) with anti-His antibodies and Coomassie staining for GST-Mex67 (bottom panel) are shown. Quantification of the signal observed for His₆-Nab2 association with GST-Mex67 is indicated below. (C) Size fractionation of purified Mex67/Mtr2, Nab2, and Yra1 alone or in complex. His₆-Nab2-, His₆-Yra1-, His₆-Mtr2/Mex67-, or His₆-Mtr2-purified proteins were loaded on S200 sizing columns alone or after complex assembly of His₆-Mtr2/Mex67 or His₆-Mtr2 with His₆-Nab2 and His₆-Yra1 as indicated on the right. (Top numbers) Collected fractions were analyzed by Western blotting with antibodies specific for Nab2, Yra1, Mex67, and Histidine to detect Mtr2.

Yra1 is ubiquitinated by Tom1. (A) HA-YRA1 cDNA-shuffled (lanes 2,3) and Δtom1 HA-YRA1 cDNA-shuffled (lane 4) strains containing a P_CUP1∷HIS₆-ubiquitin plasmid (+) or an empty vector (−). His₆-Ub-conjugated forms purified on nickel column (Ni⁺ column) were examined with their corresponding total input extracts by Western blotting with anti-His, anti-HA (Yra1), anti-Nab2, and anti-Mex67 antibodies. (Lane 2) The empty vector allowed us to control for background binding to the nickel column. (B) Tom1 does not affect Yra1 turnover. Wild-type (wt) and Δtom1 cells were transformed with a plasmid expressing HA-Yra1 from a galactose (GAL)-inducible promoter. Cells were exponentially grown in galactose. At time 0, cultures were shifted to 37°C to exacerbate the potential instability, and HA-Yra1 expression was shut off by the addition of 2% glucose. Yra1 turnover was followed by collecting protein samples at the indicated time points in glucose and Western blotting with antibodies against HA or the C-terminal domain of RNA polymerase II (RNA Pol II) to confirm equal loading. (C) Yra1 domain organization and binding partners, and position of the 22 lysines (black dots). (D) Yra1 ubiquitination in Yra1 wild-type or KR mutant strains. His₆-Ub-conjugated forms of HA-Yra1 from YRA1-shuffled (lanes 2,3,5,7) or Δtom1 YRA1-shuffled (lanes 4,6,8) strains expressing the indicated wild-type and Yra1 KR mutant plasmids were purified and analyzed as in A.

Yra1 ubiquitination is required for efficient mRNA export. (A) Loss of Tom1 interferes with mRNA export. (Left panel) Indicated strains were grown at 25°C, shifted for 3 h to 37°C, and analyzed for poly(A)⁺ RNA distribution with an oligo (dT)₅₀ probe. (Right panel) Cells were induced with galactose for 30 min at 30°C, and GAL1 mRNA distribution was analyzed by FISH using specific probes. DAPI staining or FISH/DAPI overlays are shown below each panel to localize the RNA signal with respect to the nucleus. (B) Loss of Yra1 ubiquitination interferes with mRNA export. Yra1 wild type and Yra1 KR9–22 cells were grown at 25°C, shifted for 1 h to 37°C, and analyzed for poly(A)⁺ RNA distribution with an oligo (dT)₅₀ probe. (C) Overexpression of Nab2 and Mex67 suppresses the Yra1 ubiquitination defect. Yra1 KR9–22 cells transformed with high-copy NAB2, MEX67 plasmids, or empty vector were grown at 25°C and analyzed for poly(A)⁺ RNA distribution as in B. (D) Quantification of FISH analyses: The average percent of cells with an mRNA export defect was determined by examining at least 100 DAPI-stained cells from three independent FISH experiments (except for wild type and Δtom1, the values of which correspond to one experiment). Note that wild type is W303, the corresponding wild type for Δtom1, while Yra1 wild type is YRA1-shuffled + YRA1 wild-type cDNA, the corresponding wild-type for KR9–22.

Nab2 and Yra1 are part of the same mRNA export pathway. (A) FISH analysis of GAL1 mRNA in the indicated cells induced with galactose for 30 min at 30°C. DAPI staining or FISH/DAPI overlays are shown below each panel to localize the RNA signal with respect to the nucleus. (B) Δyra1 is bypassed by overexpressing NAB2 or MEX67. The Yra1 shuffle strain transformed with the indicated plasmids was spotted as 10-fold serial dilutions onto 5-FOA to select against the wild-type pURA3-YRA1 plasmid, or onto a selective plate to control for cell number (last two dilutions only). (2μ) High-copy plasmids.

Yra1 ubiquitination reduces interaction with mRNP components. (A) The Yra1 KR9–22 lysine mutant increases the association of Yra1 with nuclear mRNPs. Total extracts (Input) or IgG-Sepharose-purified extracts (IP) from Cbp20-TAP-tagged or nontagged (No tag) strains expressing wild-type or KR9–22 mutant Yra1 were analyzed by Western blotting with anti-Nab2, anti-Mex67, and anti-HA antibodies to detect Yra1. Bead-associated Cbp20-TAP was revealed with an anti-ProtA antibody, and served as control for equal amounts of mRNP pulled down. (B) Tom1-mediated ubiquitination of Yra1 induces Yra1 release from Nab2–Mex67 complexes. Total extracts prepared from Nab2 ProtA-tagged or nontagged (no tag) strains transformed with Ub^K48R,G76A or empty vector (−) were purified on IgG beads. Western blotting of total extract (Input) or high-salt eluted proteins (IP) were examined with antibodies against Mex67, Yra1, and ProtA. Quantification of the signal observed in the Nab2-ProtA immunoprecipitate for Mex67 and Yra1 (normalized to the signal in wild-type cells transformed with empty vector) is indicated below.

Yra1 ubiquitination may be part of an mRNA export surveillance mechanism associated with NPCs. (A) Loss of Mlp2 suppresses the Yra1 KR mutant temperature-sensitive phenotypes. Tenfold serial dilutions of the indicated YRA1-shuffled strains were spotted onto YEPD plates and grown at the indicated temperatures. (B) Loss of Mlp2 suppresses the Δtom1 growth defect. Indicated strains were analyzed as in A. (C) Loss of Rrp6 enhances the growth defect of Δtom1. Indicated strains were analyzed as in A. (D) Model showing the ubiquitin-dependent release of Yra1 from Mlp-retained mRNP complexes, allowing their nuclear exit. Ubiquitination is mediated by Tom1 and another still unidentified E3 ligase (not shown).