ASH1 mRNA and IST2 mRNA are delocalized in cells expressing She2p mutants N36S and R63K. (A) Strain YLM851 was transformed with plasmids YCplac111 (vector), pRL199 (She2p-wt), pRL322 (She2p-R63K), or pRL324 (She2p-N36S). Transformants were patched on low adenine media devoid of leucine or streaked for single colonies on media devoid of leucine and arginine in the (+) presence and (−) absence of canavanine. (B) Strain YLM777 was transformed with plasmid C3431 (YEplac195/ASH1) and pRL199, pRL322, or pRL324. Transformants were grown in media devoid of leucine and uracil. Cells were processed for in situ hybridization using Cy3-conjugated complementary DNA probes for ASH1 mRNA. Representative images for in situ hybridization, DAPI staining, and (Nom.) Nomarski optics are shown. (C) Strain YLM777 was transformed with plasmid pRL277 [pHZ18(A)-IST2] and pRL199, pRL322, or pRL324. Cells were processed for in situ hybridization using Cy3-conjugated complementary DNA probes for lacZ-IST2 mRNA.

She2p amino acid substitutions N36S and R63K do not affect the steady-state level of She2p. Yeast strain YLM777 was transformed with plasmids (lane 1) YCplac111 (vector), (lane 2) pRL199 (She2p-wt), (lane 3) pRL324 (She2p-N36S), or (lane 4) pRL322 (She2p-R63K). Transformants were grown in media devoid of leucine, and cell lysates were prepared. Cell lysates were used for Western blotting with anti-myc monoclonal antibody or anti-α-tubulin antibody.

She2p mutants N36S and R36K are able to interact with She3p in vitro and in vivo. (A) In vitro GST pull-down assays were performed with 1 μg of (lane 1) GST, (lane 2) GST-She2p-wt, (lane 3) GST-She2p-N36S, or (lane 4) GST-She2p-R63K incubated with a C-terminal domain of She3p prepared by coupled in vitro transcription/translation. (B) She2p mutants N36S and R63K are able to interact with She3p by the two-hybrid assay. Strain PJ69-4a was transformed with the following combinations of plasmids: pGBDU-c1 (vector)/pACT2 (vector), pRL418 (pGBDU-c1-She2p-wt)/pACT2, pGBDU-c1/pEP13 (pACT2-She3p), pRL418/pEP13, pRL419 (pGBDU-c1-She2p-N36S)/pEP13, and pRL431 (pGBDU-c1-She2p-R63K)/pEP13. Transformants were grown in liquid culture and processed for β-galactosidase assays. (C) Characterization of the anti-She2p rabbit antiserum. Extracts were prepared from yeast strains YLM777 transformed with (lane 1) YCplac111 (vector), (lane 2) pRL453 (YCplac111-She2p), and (lane 3) pRL199 (YCplac111-She2p-myc₆). Transformants of YLM777 were grown in synthetic media devoid of leucine. Lysates were prepared and used for Western blotting with anti-She2p rabbit antiserum. In addition, 5 ng of purified (lane 4) GST, (lane 5) GST-She2p-wt, (lane 6) GST-She2p-N36S, and (lane 7) GST-She2p-R63K were analyzed by Western blot with anti-She2p rabbit antiserum. The bands corresponding to She2p, She2-myc₆, and GST-She2p are indicated by the arrows. (D) She2p mutants N36S and R63K associate with full-length endogenous She3p in vivo. Yeast strain YLM1320 was transformed with the following combinations of plasmids: (lane 1) YCplac22 (vector)/pRL453 (YCplac111-She2p-wt), (lane 2) pRL460 (YCplac22-She3p-myc₆)/pRL453, (lane 3) pRL460/YCplac111 (vector), (lane 4) pRL460/pRL462 (YCplac111-She2p-N36S), or (lane 5) pRL460/pRL463 (YCplac111-She2p-R63K). Transformants were grown in synthetic media devoid of leucine as well as tryptophan, and lysates were prepared. An aliquot of the total lysate was analyzed by Western blot for She3p-myc₆ and She2p (total). She3p-myc₆ was immunoprecipitated from the lysate using anti-myc antibody, and the immunoprecipitates were analyzed by Western blot for the presence of She3p-myc₆ and She2p (IP). She2p was detected in the immunoprecipitates using anti-She2 rabbit antiserum. Subsequently, the Western blot was stripped and reprobed with anti-myc to detect She3p-myc₆.

She2p mutants N36S and R63K are defective for ASH1 and IST2 mRNA-binding activity. (A) The UV cross-linking assay was performed by incubating (lane 1) 0.25 μg of GST-She2p-wt, (lanes 2,3) 0.5 μg of GST-She2p-wt, (lanes 4,5) 5.0 μg of GST-She2p-N36S, or (lanes 6,7) 5.0 μg of GST-She2p-R63K with 1 ng of (lanes 1,2,4,6) ³²P-labeled E3 RNA or (lanes 3,5,7) MS2 RNA. The arrow indicates the position of the GST-She2p/RNA complex. (B) Apparent gel shift of She2p with E3 RNA is not caused by RNA–protein complex formation. (Lane 1) Trace (1 nM) labeled E3 RNA combined with (lane 2) mobility shift buffer (MSB) produces an apparent mobility shift in the absence of protein. Addition of (lanes 3–6) 125 nM She2p with (lanes 4–6) increasing molar equivalents of unlabeled E3 competitor RNA results in an increase, not a decrease, in the amount of the shifted species, and is consistent with the formation of a base-paired RNA duplex, not an RNA–protein complex. (C) She2p RNA-binding activity for mutants N36S and R63K as measured by three-hybrid assay. Strain YLM585 was transformed with the following combinations of plasmids: pIIIA-MS2–2(vector)/pGAD-c1 (vector), pIIIA-MS2-2/pRL445 (pGAD-c1-Shep-wt), pRL80 (pIIIA-MS2-2-E3)/pRL445, pRL80/pRL441 (pGAD-c1-She2p-N36S), and pRL80/pRL442 (pGAD-c1-She2p-R63K). Transformants were grown in liquid culture and processed for β-galactosidase assay. (D) She2p RNA-binding activity for She2p mutants N36S and R63K as measured by IP/RT-PCR. Yeast strain YLM777 was transformed with plasmid C3431 (YEplac195/ASH1) and plasmid (lanes 1,2) pRL199 (She2-myc₆-wt), (lane 3) pRL324 (She2-myc₆-N36S), or (lane 4) pRL322 (She2p-myc₆-R63K). Transformants were grown in synthetic media devoid of leucine and uracil; lysates were prepared and immunoprecipitations were performed with anti-myc. Immunoprecipitations were subsequently used for RT-PCR reactions with primers specific for ASH1 mRNA or IST2 mRNA. RT-PCR reactions corresponding to an aliquot of the lysates prior to immunoprecipitation (total) are shown as well as reactions corresponding to the immunoprecipitation reactions with and without RT (+RT and −RT). Also, an aliquot of the immunoprecipitation was analyzed by Western blot using anti-myc.

R43, R44, and R52 are required for She2p association with ASH1 mRNA. (A) Amino acid sequence for She2p with amino acids N36, R43, R44, R49, R52, and R63 indicated by the black arrowheads. The black rectangle indicates the region of She2p corresponding to the predicted leucine zipper motif. (B) She2p RNA-binding activity for mutants R43K, R43A, R44K, R44A, R49K, R49A, R52K, R52A, and R63A as measured by three-hybrid assay. Strain YLM585 was transformed with the following combinations of plasmids: pRL80 (pIIIA-MS2-2-E3)/pRL445 (pGAD-c1-She2p-wt), pRL80/pRL552 (pGAD-c1-She2p-R43K), pRL80/pRL669 (pGAD-c1-She2p-R43A), pRL80/pRL553 (pGAD-c1-She2p-R44K), pRL80/pRL670 (pGAD-c1-She2p-R44A), pRL80/pRL554 (pGAD-c1-She2p-R49K), pRL80/pRL671 (pGAD-c1-She2p-R49A), pRL80/pRL555 (pGAD-c1-She2p-R52K), pRL80/pRL672 (pGAD-c1-She2p-R52A), and pRL80/pRL673 (pGAD-c1-She2p-R63A). Transformants were grown in liquid culture and processed for β-galactosidase assays.

(A) The intracellular distribution of She2p mutants N36S and R63K is not altered. Representative images for the intracellular distribution of She2p-myc₆. Yeast strain YLM777 was transformed with pRL453 (control), pRL199 (She2p-myc₆-wt), pRL324 (She2p-myc₆-N36S), or pRL322 (She2p-myc₆-R63K). Transformants were grown in synthetic media minus leucine; subsequently, the cells were fixed and processed for immunofluorescence using anti-myc. (B) She2p does not accumulate in the nucleus in the absence of mRNA export. Yeast strain YLM1769 was transformed with the following combinations of plasmids: pRL718 (pESC-URA-ASH1)/YCplac22 (vector), pRL718/pRL676 (YCplac22-She2p-myc₆-wt), pRL718/pRL677 (YCplac22-She2p-myc₆-N36S), and pRL718/pRL678 (YCplac22-She2p-myc₆-R63K). Transformants were grown at the permissive and nonpermissive temperatures as described in Materials and Methods, Temperature Shift Experiment. For immunofluorescence, anti-myc antibody was used and ASH1 mRNA was detected by FISH.

The asymmetric distribution of Myo4p and She3p in anaphase cells is dependent on She2p mRNA-binding activity, but formation of the Myo4p/She3p complex is independent of She2p RNA-binding activity. (A) Representative images for the intracellular distribution of Myo4p-myc₁₃ in cells devoid of She2p, expressing wild-type She2p, She2p mutant N36S, and She2p mutant R63K. Strain YLM1309 was transformed with plasmid YCplac111 (vector), pRL453 (YCplac111-She2p-wt), pRL462 (YCplac111-She2p-N36S), or pRL463 (YCplac111-She2p-R63K). Transformants were grown in synthetic media minus leucine; subsequently, the cells were fixed and processed for immunofluorescence using anti-myc. Then 100 anaphase cells with signal corresponding to Myo4p were counted; below each panel, the percentage of anaphase cells with asymmetrically sorted Myo4p is indicated. (B) Representative images for the intracellular distribution of She3p-myc₁₃ in cells devoid of She2p, expressing wild-type She2p, She2p mutant N36S, or She2p mutant R63K. Strain YLM1368 was transformed and processed for immunofluorescence identically to strain YLM1309 described above. Then 100 anaphase cells with signal corresponding to She3p were counted; below each panel, the percentage of anaphase cells with asymmetrically sorted She3p is indicated. (C) The Myo4p/She3p complex can form in the absence of She2p RNA-binding activity. Strain YLM921 was transformed with plasmids (lane 1) pRL460 (YCplac22-She3p-myc₆)/pRL453 (YCplac111-She2p-wt). Yeast strain YLM1320 was transformed with the following combinations of plasmids: (lane 2) vector (YCplac22)/pRL453, (lane 3) pRL460/pRL453, (lane 4) pRL460/YCplac111 (vector), (lane 5) pRL460/pRL462 (YCplac111-She2p-N36S), or (lane 6) pRL460/pRL463 (YCplac111-She2p-R63K). Transformants were grown in synthetic media minus leucine and tryptophan. Cells were harvested, lysates were prepared, and an aliquot of the total lysate was saved for Western blotting. She3p-myc₆ was immunoprecipitated from the remaining portion of each lysate with anti-myc. The yeast lysates (total) and immunoprecipitates were analyzed by Western blot for the presence of She3p-myc₆ and Myo4p-3HA.