Visualization of U1A-ASH1 RNA in wild-type and sheΔ cells. (a) A diagram of the constructs used to visualize and immunoprecipitate Ash1 mRNA. The RNA-binding construct (U1Ap-GFP) consisted of the amino terminus of U1A fused to GFP(S65T) for visualization, glutathione S-transferase for possible use in affinity purification, and a nuclear localization sequence (N) to lower the amount of cytoplasmic fluorescence. This construct was expressed from the constitutive glycerol phosphate dehydrogenase (GPD) promoter. To provide a binding site for U1Ap-GFP, four repeats of the U1A 3′ UTR were introduced just downstream of the start codon of either the entire coding region of ASH1 and ≈500 bp of its 3′ UTR (U1A_tag-ASH1) or the entire coding region of ADH2 and ≈500 bp of its 3′ UTR (U1A_tag-ADH2). Both of these constructs were expressed from the inducible galactose promoter. (b) Cells expressing U1Ap-GFP and U1A_tag-ASH1 contain a single large particle at the distal tip of the bud, whereas cells expressing U1Ap-GFP and U1A_tag-ADH2 display an increase in cytoplasmic fluorescence but do not contain any particles. (c) sheΔ cells contain mislocalized U1A-ASH1 RNA particles. myo4Δ and she3Δ cells contain multiple particles that are distributed throughout the mother cell, whereas bni1Δ cells contain a single particle that is localized to the neck. she2Δ and she4Δ cells displayed a similar GFP fluorescence pattern as myo4Δ and she3Δ cells (not shown).

She2p, She3p, and Myo4p colocalize with the U1A-ASH1 RNA particle. Thirteen repeats of the c-myc peptide sequence were inserted at the C terminus of SHE2, SHE3, MYO4, SHE4, and BNI1 in wild-type cells (w303). All samples express both U1Ap-GFP and U1A_tag-ASH1. In untagged cells, the GFP fluorescence from the U1A-ASH1 RNA particle is visible at the distal tip of the bud but no staining is detected with the anti-myc antibody. In SHE2-MYC, SHE3-MYC, and MYO4-MYC cells, the GFP fluorescence from the U1A-ASH1 RNA particle colocalizes with the anti-myc immunofluorescence. In SHE4-MYC and BNI1-MYC cells, the U1A-ASH1 RNA GFP particle is visible at the distal tip of the bud but does not colocalize with the anti-myc fluorescence.

Immunoprecipitation of U1A-ASH1 RNA reveals an association between Ash1 mRNA and Myo4p, She2p, and She3p. Extracts were prepared from SHE-MYC cells expressing either U1Ap-GFP and U1A_tag-ASH1 or U1Ap-GFP and U1A_tag-ADH2. Extracts were incubated with anti-GFP antibodies coupled to protein A agarose. Immunoprecipitates were analyzed for the She proteins by Western blot and for U1A-ASH1 RNA and U1A-ADH2 RNA by Northern analysis. The Western blot was quantitated with National Institutes of Health image. The -fold increase of each She protein in U1A-ASH1 RNA immunoprecipitates compared with U1A-ADH2 RNA immunoprecipitates is Myo4p, 1.5; She2p, 1.5; She3p, 3.3; She4p, 1.1; and Bni1p, 1.1.

Analysis of the interactions between the She proteins and Ash1 mRNA. (a) She2p, She3p, and Myo4p associate with Ash1 mRNA. She-myc proteins were immunoprecipitated and analyzed for Ash1 mRNA and Adh2 mRNA by Northern blot. (b) Dependence on the other SHE genes for the interaction between She2p, She3p, Myo4p, and Ash1 mRNA. SHE2, SHE3, and MYO4 were c-myc tagged in sheΔ cells, and the proteins were immunoprecipitated and analyzed for Ash1 mRNA by Northern blot. (c) She3p and Myo4p form a RNase-insensitive complex. Cells were HA-tagged at MYO4 and c-myc tagged at SHE3. Extracts were prepared from MYO4-HA cells and MYO4-HA/SHE3-MYC cells and incubated with anti-myc antibodies coupled to protein G Sepharose or protein G Sepharose alone as a control. Extracts prepared from MYO4-HA/SHE3-MYC cells were either mock treated or incubated with RNase A. The immunoprecipitates were analyzed for the amount of Myo4p by Western blot.

A model of Ash1 mRNA localization. She2p binds Ash1 mRNA which then allows the She3p/Myo4p complex to bind. She3p mediates the interaction between Ash1 mRNA and Myo4p. Myo4p transports the Ash1 ribonucleoprotein complex along actin filaments to the distal tip of the daughter cells.