She2p-myc interacts cotranscriptionally with bud-localized mRNAs. (A) Diagram of ASH1 and IST2 genes, and the relative position of the amplicons used for each gene. Dark boxes in ASH1 and IST2 correspond to the localization elements. In the ASH1 gene, amplicon Pro spans a region 500 base pairs (bp) upstream of the start codon, while the amplicon Nt is upstream of the start codon. Amplicons E1, E2A, and E3 overlap with the 5′ half of the corresponding localization element. (B) She2p-myc immunoprecipitates the endogenous ASH1 coding sequence near the localization elements in an RNA-dependent manner. She2p-myc ChIP was performed using an anti-myc antibody, followed by PCR amplification of amplicons specific to endogenous ASH1 (Pro, Nt, E1, E2A, and E3). Chromatin was treated (+RNase) or not with RNase A prior immunoprecipitation. (C) Transcription-dependent interaction between She2p-myc and a galactose-inducible ASH1 gene. She2p-myc ChIP was performed using an anti-myc antibody, followed by PCR amplification of amplicons specific to the galactose-induced (galactose) or repressed (glucose) ASH1 gene (E1 and E2A). Chromatin was treated (+RNase) or not with RNase A prior immunoprecipitation. (D) Kinetics of She2p-myc recruitment on the ASH1 gene, and interaction with ASH1 mRNA. Interaction of She2p-myc with the ASH1 gene was determined by ChIP, followed by qPCR amplification of amplicon E1. Interaction of She2p-myc with ASH1 mRNA was determined by immunoprecipitation followed by qRT–PCR. ASH1 mRNA enrichment was normalized to IST2 mRNA levels. (Left Y-axis) ASH1 amplicon enrichment by ChIP. (Right Y-axis) ASH1 mRNA enrichment by RT–PCR. (E) She2p-myc immunoprecipitates the IST2 coding sequence near the localization element. She2p-myc ChIP was performed using an anti-myc antibody, followed by PCR amplification of the amplicon specific to endogenous IST2 (Nt or Ct). Chromatin was treated (+RNase) or not with RNase A prior immunoprecipitation. The data are presented as the mean ± SEM (N ≥ 3).

She2p-myc interacts with the elongating form of RNA pol II in vivo. (A) Rpb1p coimmunoprecipitates with She2p-myc. She2p-myc was immunoprecipitated with an anti-myc antibody (9E10), followed by detection of Rpb1p by Western blot using the 8WG16 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (IP + RNase) yeast extract treated with RNase prior to immunoprecipitation; (CTL) strain K699 with untagged She2p. Pgk1 was used as a negative control. (B) She2p-myc coimmunoprecipitates with Rpb1p. Rpb1p was immunoprecipitated with the 8WG16 antibody, followed by detection of She2p-myc by Western blot using the 9E10 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (IP + RNase) yeast extract treated with RNase prior to immunoprecipitation; (CTL) strain K699 with untagged She2p. Pgk1 was used as negative control. (C) Rpb1p interacts with She2R63K-myc, a mutant defective in RNA binding. She263K-myc was immunoprecipitated with an anti-myc antibody (9E10), followed by detection of Rpb1p by Western blot using the 8WG16 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (CTL) strain K699 with untagged She2R63K. Pgk1 was used as a negative control. (D) The elongating form of Rpb1p coimmunoprecipitates with She2p-myc. She2p-myc was immunoprecipitated with an anti-myc antibody (9E10), followed by detection of hypophosphorylated (8WG16), Ser5-phosphorylated (H5), or Ser-2-phosphorylated (H14) Rpb1p by Western blot. (Input) Total yeast extract; (IP) immunoprecipitate; (IP + RNase) yeast extract treated with RNase prior to immunoprecipitation; (CTL) strain K699 with untagged She2p. Pgk1 was used as negative control. (E) She2p-myc coimmunoprecipitates with Ser5-phosphorylated Rpb1p. Ser5-phosphorylated Rpb1p was immunoprecipitated with the H5 antibody, followed by detection of She2p-myc by Western blot using the 9E10 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (IP + RNase) yeast extract treated with RNase prior to immunoprecipitation; (CTL) strain K699 with untagged She2p. Pgk1 was used as negative control. (F) She2p-myc coimmunoprecipitates with Ser2-phosphorylated Rpb1p. Ser2-phosphorylated Rpb1p was immunoprecipitated with the H14 antibody, followed by detection of She2p-myc by Western blot using the 9E10 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (IP + RNase) yeast extract treated with RNase prior to immunoprecipitation; (CTL) strain K699 with untagged She2p. Pgk1 was used as a negative control.

She2p-myc interacts with RNA pol II via the transcription elongation factor Spt4–Spt5. (A) Deletion of SPT4 decreases the interaction between She2p-myc and Rpb1p in vivo. Rpb1p was immunoprecipitated with the 8WG16 antibody, followed by detection of She2p-myc by Western blot using the 9E10 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (CTL) yeast extract treated with beads only (no antibody). The ratio She2p/Rpb1p reflects the mean ± SEM (N = 3). (B) Temperature-sensitive mutations in SPT5 decrease the interaction between She2p-myc and Rpb1p in vivo. Rpb1p was immunoprecipitated with 8WG16 antibody, followed by detection of She2p-myc by Western blot using the 9E10 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (CTL) yeast extract treated with beads only (no antibody). The ratio She2p/Rpb1p reflects the mean ± SEM (N = 3). (C) She2p-myc coimmunoprecipitates with Spt4-TAP and Spt5-TAP. Spt4-TAP or Spt5-TAP was immunoprecipitated with IgG, followed by detection of She2p-myc by Western blot using the 9E10 antibody. (Input) Total yeast extract; (IP) immunoprecipitate; (CTL) yeast extract treated with beads only (no antibody). Pgk1 was used as a negative control. (D) Recombinant GST-She2p interacts with yeast-purified Spt4–Spt5 complex in vitro. The Spt4–Spt5 complex was purified from yeast either as a Spt4-TAP/Spt5 complex (Spt4-TAP) or Spt4/Spt5-TAP complex (Spt5-TAP). (Input) Purified recombinant proteins.

Cotranscriptional interaction of She2p-myc with ASH1 depends on the transcription elongation factor Spt4–Spt5. (A) Mutation in SPT4 or SPT5 decreases She2p-myc ChIP of the endogenous ASH1 gene. She2p-myc ChIP was performed using an anti-myc antibody, followed by qPCR amplification of amplicons specific to endogenous ASH1 (Pro, Nt, E2A, and E3) in isogenic wild-type (WT; BY4741 SHE2-MYC), paf1, and spt4 strains, or in isogenic wild-type (WT; FY119 SHE2-MYC) and spt5-194 mutant strains at 25°C or 37°C. (B) Mutations in SPT4 and SPT5 do not affect Rpb1p ChIP on the ASH1 gene. Experiments were performed as in A, with the exception that the 8WG16 antibody was used for ChIP. (C) RNase treatment reduces She2p-myc ChIP in the spt4 strain. ChIP was performed as in A, with the addition (+) or not (−) of RNase A prior to immunoprecipitation. (D) RNase treatment reduces She2p-myc ChIP in the spt5-194 mutant strain. ChIP was performed as in A, with the addition (+) or not (−) of RNase A prior to immunoprecipitation. The data are presented as the mean ± SEM (N = 3).

Spt4–Spt5 is required for proper ASH1 mRNA localization and Ash1p sorting. (A) Phenotypes of ASH1 mRNA localization as detected by FISH. (Localized) ASH1 mRNA localized at the bud tip; (Bud-localized) ASH1 mRNA filling the entire bud. (Delocalized) ASH1 mRNA in both the bud and the mother cell. Bar, 2 μm. (B) Scores on ASH1 mRNA localization phenotypes from wild-type (WT), paf1, and spt4 strains. (C) Scores on ASH1 mRNA localization phenotypes from wild-type (WT) and spt5-4 and spt5-194 mutant strains at 25°C or 37°C. (D) Yeast genetic assay for Ash1p asymmetric distribution. Tenfold dilutions of exponentially growing K5547 (HO-ADE2, she2) transformed with either YCP22-She2-myc (SHE2/SPT4) or YCP22 empty (she2/SPT4), or strain K5547 spt4∷KAN transformed with YCP22-She2-myc (SHE2/spt4) were spotted on plates lacking tryptophan (−Trp) or lacking tryptophan and adenine (−Trp −Ade), and were incubated at 30°C.

She2p-myc interacts cotranscriptionally with genes coding for both nonlocalized and bud-localized mRNAs. (A) Diagram of EAR1, PGK1, PMA1, ACT1, ASC1, and FBA1 genes, and the relative position of the amplicons used for each gene. Dark boxes in EAR1 correspond to localization elements. The gray box in ACT1 and ASC1 corresponds to their introns. For all ChIP experiments, RNA pol II (Rpb1p) and She2p-myc ChIPs were performed using 8WG16 and anti-myc antibodies, respectively. qPCR amplification of amplicons specific to endogenous genes was performed on immunoprecipitated chromatin. Chromatin was treated (+RNase) or not with RNase A prior to immunoprecipitation. All ChIPs were performed using strain W303-SHE2-MYC, with the exception of E. (B) She2p-myc is not associated with genes coding for tRNA^CUU, tRNA^CUG, and U6 snRNA, which are transcribed by RNA pol III. (C) She2p-myc immunoprecipitates the ASH1, IST2, and EAR1 genes, which encode bud-localized mRNAs. (*) P < 0.001. (D) She2p-myc immunoprecipitates the ACT1, PMA1, FBA1, PGK1, and ASC1 genes, which do not encode for bud-localized/She2p-associated mRNAs. (E) An RNA-binding-defective mutant of She2p, She2R63K, still associates with pol II transcribed genes by ChIP. Experiments were performed using strain K699 she2 strain transformed with YCP22-She2-myc or YCP22-She2R63K-myc. The data are presented as the mean ± SEM (N = 3).

RNA-dependent recruitment of She2p on a gene requires a zipcode. (A) The presence of a zipcode in the LacZ gene results in RNase-sensitive association of She2p-myc by ChIP. Yeast strains with integrated galactose-inducible LacZ or LacZ-E3 genes were grown under repressed (glucose) or activated (galactose) transcription. Chromatin was treated (+RNase) or not with RNaseA prior to She2p-myc ChIP. The data are presented as the mean ± SEM (N = 3). (B) Model for cotranscriptional recruitment of She2p to bud-localized mRNAs. (Left) She2p interacts with RNA pol II via the Spt4–Spt5 transcription elongation factor. Association of She2p with the TEC (middle) allows the transfer of She2p to a zipcode in the nascent mRNA as it emerges from the RNA polymerase (right).