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• W2104514904 abstract "Article1 March 2002free access The Khd1 protein, which has three KH RNA-binding motifs, is required for proper localization of ASH1 mRNA in yeast Kenji Irie Kenji Irie Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, Japan Departments of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Tomofumi Tadauchi Tomofumi Tadauchi Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, Japan Search for more papers by this author Peter A. Takizawa Peter A. Takizawa Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Ronald D. Vale Ronald D. Vale Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Kunihiro Matsumoto Corresponding Author Kunihiro Matsumoto Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, Japan Search for more papers by this author Ira Herskowitz Ira Herskowitz Departments of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Kenji Irie Kenji Irie Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, Japan Departments of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Tomofumi Tadauchi Tomofumi Tadauchi Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, Japan Search for more papers by this author Peter A. Takizawa Peter A. Takizawa Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Ronald D. Vale Ronald D. Vale Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Kunihiro Matsumoto Corresponding Author Kunihiro Matsumoto Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, Japan Search for more papers by this author Ira Herskowitz Ira Herskowitz Departments of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA Search for more papers by this author Author Information Kenji Irie1,2,3, Tomofumi Tadauchi1,2, Peter A. Takizawa4, Ronald D. Vale4, Kunihiro Matsumoto 1,2 and Ira Herskowitz3 1Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya, 464-8602 Japan 2CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya, Japan 3Departments of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143-0448 USA 4Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94143-0448 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1158-1167https://doi.org/10.1093/emboj/21.5.1158 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info RNA localization is a widespread mechanism for achieving localized protein synthesis. In Saccharo myces cerevisiae, Ash1 is a specific repressor of transcription that localizes asymmetrically to the daughter cell nucleus through the localization of ASH1 mRNA to the distal tip of the daughter cell. This localization depends on the actin cytoskeleton and five She proteins, one of which is a type V myosin motor, Myo4. We show here that a novel RNA-binding protein, Khd1 (KH-domain protein 1), is required for efficient localization of ASH1 mRNA to the distal tip of the daughter cell. Visualization of ASH1 mRNA in vivo using GFP-tagged RNA demonstrated that Khd1 associates with the N element, a cis-acting localization sequence within the ASH1 mRNA. Co-immunoprecipitation studies also indicated that Khd1 associates with ASH1 mRNA through the N element. A khd1Δ mutation exacerbates the phenotype of a weak myo4 mutation, whereas overexpression of KHD1 decreases the concentration of Ash1 protein and restores HO expression to she mutants. These results suggest that Khd1 may function in the linkage between ASH1 mRNA localization and its translation. Introduction The asymmetric distribution of proteins is vital to cellular function and cell fate determination. One mechanism for achieving asymmetric distribution of a protein is by localizing its mRNA to a distinct site within the cell. Localization of mRNAs is specified by sequences generally found in the 3′ untranslated region (3′ UTR) of the mRNA, and is mediated by cytoskeletal filaments that are required for transport and subsequent anchoring of the mRNA at its final destination (Wilhelm and Vale, 1993; St Johnston, 1995; Nasmyth and Jansen, 1997; Oleynikov and Singer, 1998). The transport, anchoring and translational regulation of localized transcripts are governed by proteins that form large ribonucleoprotein complexes with the mRNAs (Wilhelm and Vale, 1993; Hazelrigg, 1998). The asymmetric distribution of Ash1 in the budding yeast Saccharomyces cerevisiae provides an excellent opportunity to study the asymmetric segregation of cell fate determinants resulting from mRNA localization. Ash1 is a cell-type specific transcriptional repressor that determines proper mating-type switching by differentially regulating expression of the HO endonuclease (Bobola et al., 1996; Sil and Herskowitz, 1996). Ash1 is found in the nucleus of daughter cells, where it represses HO transcription and ultimately prevents mating-type switching in these cells (Bobola et al., 1996; Sil and Herskowitz, 1996). This transcriptional regulation of HO expression restricts mating-type switching to mother cells (Nasmyth, 1983; Herskowitz, 1988). The asymmetric distribution of Ash1 to daughter cell nuclei is a result of the localization of ASH1 mRNA to the distal tips of daughter cells (Long et al., 1997; Takizawa et al., 1997). Five genes have been identified that are required for ASH1 mRNA localization; SHE1–SHE5 (Jansen et al., 1996; Long et al., 1997; Takizawa et al., 1997). SHE1 encodes a type V myosin motor, Myo4, which co-localizes with ASH1 mRNA at the tip of daughter cells (Haarer et al., 1994; Bertrand et al., 1998; Munchow et al., 1999; Takizawa and Vale, 2000). Using a live-cell assay, particles containing Myo4 and ASH1 mRNA were observed to move rapidly from mother cells to daughter cells, suggesting that Myo4 plays a direct role in transporting ASH1 mRNA to the bud tip (Bertrand et al., 1998; Beach et al., 1999; Takizawa and Vale, 2000). Immunoprecipitation experiments have revealed that Myo4 associates with ASH1 mRNA and that this association is dependent on SHE2 and SHE3 (Munchow et al., 1999; Takizawa and Vale, 2000). SHE2 encodes an RNA-binding protein that directly binds to ASH1 mRNA (Bohl et al., 2000; Long et al., 2000). The C-terminus of She3 interacts with She2, while its N-terminus interacts with Myo4 (Bohl et al., 2000; Long et al., 2000). Thus, She3 has the properties of an adapter that links Myo4 to the She2–ASH1 mRNA complex. SHE5 is identical to BNI1, which was shown to encode a protein involved in regulating the actin cytoskeleton (Jansen et al., 1996; Kohno et al., 1996; Evangelista et al., 1997). She4 is also hypothesized to be required for proper organization of the actin cytoskeleton (Jansen et al., 1996; Wendland et al., 1996). Taken together, these results suggest that ASH1 mRNA is localized to the bud tip by actomyosin-based transport. Loc1, a nuclear RNA-binding protein, is also involved in ASH1 mRNA localization (Long et al., 2001). Based on these studies, the following model for ASH1 mRNA localization has been proposed (Bohl et al., 2000; Long et al., 2000, 2001; Takizawa and Vale, 2000; Kwon and Schnapp, 2001). First, the ASH1 mRNA is identified by Loc1 in the nucleus. Secondly, the ASH1 mRNA is transported through the nuclear pores to the cytoplasm, where it binds to the cytoplasmic RNA-binding protein She2. Thirdly, the She2–ASH1 mRNA complex associates with Myo4 via the She3 adapter protein. Finally, the ASH1 mRNA–She2–She3–Myo4 complex is transported to the distal tips of daughter cells along polarized actin filaments. In cases where protein localization is determined by mRNA localization, it can be expected that translation of the mRNA would be blocked until its proper localization at the distant site. Thus, mRNA localization is likely to be tightly coupled to its translational control (Curtis et al., 1995; St Johnston, 1995; Preiss and Hentze, 1999). Indeed, several examples are known in which translational control is directly linked to protein localization. For example, in Drosophila, translation of maternal oskar mRNA is silenced during transport to the posterior pole of the oocyte and later activated when Oskar protein is required (Macdonald and Smibert, 1996). It has been shown that the protein Bruno binds to the 3′ UTR of oskar mRNA and prevents premature translation (Kim-Ha et al., 1995; Gunkel et al., 1998). It is therefore likely that additional components, such as RNA-binding proteins, contribute to efficient localization of ASH1 mRNA through regulation of its translation. During our studies on the identification and characterization of RNA-binding proteins required for ASH1 mRNA localization, we identified a previously uncharacterized yeast protein, Khd1. In this study we show that Khd1 is required for the tight anchoring of ASH1 mRNA to the distal tip of the daughter cell. Khd1 both co-localizes and physically associates with ASH1 mRNA. Over expression of Khd1 causes decreased Ash1 protein concentrations. These results suggest that Khd1 functions in the linkage between ASH1 mRNA localization and its translation. Results A putative RNA-binding protein involved in proper localization of ASH1 mRNA To identify proteins required for ASH1 mRNA localization, we carried out a systematic survey of the different candidate RNA-binding proteins and their effects on ASH1 mRNA localization. The yeast genome contains five genes, PUF1/JSN1, PUF2, PUF3, PUF4/YGL014w and PUF5/MPT5, that code for homologs of the Puf family of RNA-binding proteins (Zhang et al., 1997; Olivas and Parker, 2000; Tadauchi et al., 2001) and five genes, MER1, MSL5, PBP2, SCP160 and YBL032w, which code for proteins that contain the KH RNA-binding motif (Engebrecht and Roeder, 1990; Van Dyck et al., 1994; Abovich and Rosbash, 1997; Weber et al., 1997; Mangus et al., 1998). We constructed mutants of each of these nine genes by PCR-mediated gene disruption, except for MSL5, which is an essential gene (Abovich and Rosbash, 1997) (see Materials and methods). Disruptants of each of the nine genes were viable, although mpt5Δ and scp160Δ mutants exhibited temperature-sensitive growth at 37°C. We examined ASH1 mRNA localization in these deletions by in situ hybridization (Table I). ASH1 mRNA was partially delocalized in mpt5Δ, scp160Δ and ybl032wΔ mutants, whereas ASH1 mRNA was properly localized in the other six mutants. As YBL032w had not been characterized previously, we designated it KHD1 (KH-domain protein 1). Table 1. ASH1 mRNA localization in disruptants of genes encoding RNA-binding proteins Genotype % (n = 100) Anchored Delocalized in the bud Delocalized in mother and bud Neck Wild type 87 12 1 0 puf1Δ/jsn1Δ 85 14 1 0 puf2Δ 69 29 2 0 puf3Δ 85 15 0 0 puf4Δ 83 16 1 0 puf5Δ/mpt5Δ 22 61 16 1 scp160Δ 23 61 16 1 pbp2Δ 79 20 1 0 khd1Δ/ybl032wΔ 53 40 7 0 mer1Δ 78 20 2 0 Anchored: tightly localized ASH1 mRNA at the distal tip; delocalized in the bud: delocalized ASH1 mRNA confined to the bud; delocalized in mother and bud: ASH1 mRNA in both mother cell and bud; neck: ASH1 mRNA at the bud neck. To assess whether Khd1, Scp160 and/or Mpt5 play a direct role in ASH1 mRNA localization, we analyzed whether these proteins co-localized with ASH1 mRNA using a system in which U1A-tagged ASH1 mRNA is marked with green fluorescent protein (GFP) (Takizawa and Vale, 2000). In this experimental system, cells are transformed with two plasmids, U1Ap-GFP and U1Atag-ASH1. U1Ap-GFP expresses a fusion protein of the RNA-binding domain of U1A and a variant of GFP (S65T) in which Ser65 is changed to threonine. U1Atag-ASH1 expresses ASH1 mRNA containing the U1A-binding sequence downstream of the start codon under the control of the GAL1 promoter. Cells expressing U1Ap-GFP and U1Atag-ASH1 display a single large GFP particle localized to the distal tips of daughter cells, and Myo4, She2 and She3 co-localize with this particle (Takizawa and Vale, 2000). We constructed strains harboring myc-tagged versions of Khd1, Scp160 and Mpt5 as described in Materials and methods. These tagged proteins carry 13 repeats of the c-myc peptide at the C-terminus of each protein. These strains displayed normal localization of ASH1 mRNA, indicating that the addition of the myc-tag to the proteins did not impair their function (data not shown). Khd1myc, Scp160myc and Mpt5myc strains were transformed with U1Ap-GFP and U1Atag-ASH1 plasmids and tested for co-localization of the myc-tagged proteins with the GFP signal. Khd1myc co-localized with the GFP-tagged U1Atag-ASH1 RNA particle, whereas Scp160myc and Mpt5myc did not (Figure 1). These results suggest that Khd1 has a direct role in ASH1 mRNA localization. Below, we further characterize the role of Khd1 in the regulation of ASH1 mRNA localization. Figure 1.Khd1 co-localizes with the U1Atag-ASH1 RNA particle. Thirteen repeats of the c-myc peptide sequence were inserted at the C-terminus of Khd1, Scp160 and Mpt5 in wild-type cells (10B). All samples expressed both U1Ap-GFP (pPT220) and U1Atag-ASH1 (pPT120). In untagged cells, GFP fluorescence from the U1A-ASH1 RNA particle is visible at the distal tip of the bud (arrowhead), but no staining was detected with the anti-myc antibody. In YKEN203 (Khd1myc) cells, GFP fluorescence from the U1A-ASH1 RNA particle co-localizes with anti-myc immunofluorescence (arrow). In YKEN202 (Scp160myc) and YKEN201 (Mpt5myc) cells, the U1A-ASH1 RNA GFP particle is visible at the distal tip of the bud (arrowhead) but does not co-localize with anti-myc fluorescence. Download figure Download PowerPoint The N element of ASH1 mRNA is responsible for co-localization with Khd1 ASH1 mRNA contains three or four cis-acting localization elements: N, C and U (Gonzalez et al., 1999), or E1, E2AB and E3 (Chartrand et al., 1999) (Figure 2). Each of these elements is sufficient for localization of a heterologous reporter mRNA to daughter cells. Two regions (N, C; E1, E2AB) are located in the ASH1 open reading frame (ORF), whereas the U and E3 regions are located in the 3′ UTR. To determine which regions are responsible for the co-localization of ASH1 mRNA with Khd1, we constructed U1A-tagged versions of each element, U1Atag-N, -C and -U, in addition to U1Atag-Full, which contains all of these elements (Figure 2). Each of these constructs produced a bright particle in buds when co-expressed with U1Ap-GFP, indicating that each of the three RNA elements is sufficient to form a particle and localize to buds in the U1Atag constructs that we used (Figure 3). We then tested co-localization of Khd1myc, Myo4myc, She2myc and She3myc to each element (Figures 2 and 3). We found that Myo4myc, She2myc and She3myc co-localized with the GFP signals from all three derivatives of the U1Atag-ASH1 RNA particle (Figures 2 and 3). In contrast, Khd1myc co-localized with U1Atag-N but not with U1Atag-C or U1Atag-U (Figures 2 and 3). These results suggest that Khd1 may have a role different from that of Myo4, She2 and She3, which function in ASH1 mRNA transport (Bertrand et al., 1998; Munchow et al., 1999; Bohl et al., 2000; Long et al., 2000; Takizawa and Vale, 2000). Figure 2.Localization elements involved in ASH1 mRNA localization and co-localization with Myo4, She2, She3 and Khd1. ASH1 mRNA contains three or four localization elements [N, C and U in Gonzalez et al. (1999); E1, E2AB and E3 in Chartrand et al. (1999)]. U1Atag-N, U1Atag-C and U1Atag-U are U1A-tagged versions of each element. Right column indicates co-localization of Myo4myc, She2myc, She3myc and Khd1myc to GFP particles of U1A-tagged versions of U1Atag-Full, U1Atag-N, U1Atag-C and U1Atag-U. +, co-localization; −, no co-localization. Download figure Download PowerPoint Figure 3.The N element of ASH1 mRNA is responsible for co-localization of Khd1. (A) Khd1myc co-localizes with U1Atag-N but not with U1Atag-C or U1Atag-U. (B) She3myc co-localizes with U1Atag-N, U1Atag-C and U1Atag-U. Arrowhead, GFP fluorescence from the U1A-ASH1 RNA particle visible at the distal tip of the bud; arrow, anti-myc immunofluorescence that co-localizes with the GFP fluorescence from the U1A-ASH1 RNA particle. Strains used: YKEN203 (Khd1myc), 134 (She3myc). Download figure Download PowerPoint Khd1 associates with ASH1 mRNA in vivo Co-localization of ASH1 mRNA and Khd1 suggested that Khd1 is associated with ASH1 mRNA in vivo. To test this possibility, we investigated whether ASH1 mRNA co-immunoprecipitated with Khd1myc using immunoprecipitation and RT–PCR. We used myc-tagged She3 as a positive control, as Munchow et al. (1999) have shown that ASH1 mRNA co-immunoprecipitates with She3myc. Khd1myc and She3myc strains were transformed with a control plasmid and YEpASH1. Cell lysates were prepared from these strains and used for immunoprecipitation with anti-myc monoclonal antibody. The anti-myc antibody efficiently precipitated Khd1myc and She3myc proteins from yeast extracts (Figure 4A). By RT–PCR analysis of the immunoprecipitates, we detected endogenous ASH1 mRNA in immunoprecipitates from Khd1myc and She3myc strains. In contrast, we did not detect ASH1 mRNA in immunoprecipitates from the untagged strain, even when ASH1 was overexpressed (Figure 4A). The PCR product was not seen when reverse transcriptase was omitted, indicating that formation of this band is dependent on RNA (data not shown). These data indicate that Khd1 associates with ASH1 mRNA in vivo. Figure 4.Khd1 associates with ASH1 mRNA. Khd1 and She3 tagged with the myc epitope were immunoprecipitated using anti-myc antibody 9E10 (myc) or control IgG (c) as described in Materials and methods. Each immunopellet was separated on a 10% SDS–PAGE gel, blotted and probed with anti-myc antibody or anti-GFP antibody for the presence of epitope-tagged proteins (Khd1myc, She3myc or U1Ap-GFP). RNA was extracted from cell extracts (Total) and immuno precipitates (IP) and used as template for RT–PCR. (A) A 360 bp product was amplified using ASH1-specific primers. (B) PCR products of 420 and 380 bp were amplified using specific primers for U1Atag-N (N) and U1Atag-U (U), respectively. One-fifth of each reaction was separated on a 2% agarose gel and stained with ethidium bromide. (A) Lane 1, untagged (YEpASH1); lane 2, Khd1myc (YEplac181); lane 3, Khd1myc (YEpASH1); lane 4, She3myc (YEplac181); lane 5, She3myc (YEpASH1). (B) Lanes 1 and 2, Khd1myc (U1Atag-N + U1Ap-GFP); lanes 3 and 4, Khd1myc (U1Atag-U + U1Ap-GFP); lanes 5 and 6, She3myc (U1Atag-U + U1Ap-GFP). Total amounts of U1Ap-GFP were the same in each cell extract (data not shown). Strains used: 10B (untagged), YKEN203 (Khd1myc), 134 (She3myc). Download figure Download PowerPoint To examine whether the association of Khd1myc with ASH1 mRNA is mediated by the N element, Khd1myc proteins were immunoprecipitated from the Khd1myc strain co-expressing U1Atag-N or U1Atag-U, and U1Ap-GFP. By RT–PCR analysis of the immunoprecipitates with the anti-myc antibody, the U1Atag-N mRNA was detected in the immunoprecipitates (Figure 4B, lane 2). U1Ap-GFP also co-immunoprecipitated with Khd1myc, suggesting that Khd1myc makes a complex with U1Ap-GFP through the U1Atag-N mRNA. In contrast, the U1Atag-U mRNA and U1Ap-GFP did not co-immunoprecipitate with Khd1myc in the Khd1myc strains co-expressing U1Atag-U and U1Ap-GFP (Figure 4B, lane 4). As a control, when She3myc was co-expressed with U1Atag-U and U1Ap-GFP, the U1Atag-U mRNA and U1Ap-GFP were detected in the She3myc immunoprecipitates (Figure 4B, lane 6). These results support the possibility that the association of Khd1myc with ASH1 mRNA is mediated by the N element. Genetic interaction between the KHD1 and SHE genes Asymmetric expression of HO is ultimately determined by the localization of ASH1 mRNA (Bobola et al., 1996; Sil and Herskowitz, 1996; Long et al., 1997; Takizawa et al., 1997). Delocalization of ASH1 mRNA in she mutants causes a reduction in HO expression (Jansen et al., 1996; Long et al., 1997; Takizawa et al., 1997). Since the khd1Δ mutation partially affected ASH1 mRNA localization (Table I), we examined the effect of khd1Δ on HO expression using an HOp-ADE2 reporter gene to monitor expression of HO. HOp-ADE2 was constructed by replacing the ho ORF with the ADE2 ORF at the ho locus. Expression of the reporter can thus be assayed in an ade2Δ background by growth on medium lacking adenine (SC-Ade). myo4Δ and she3Δ mutants containing the HOp-ADE2 reporter failed to grow on SC-Ade plates (Figure 5B), demonstrating that inactivation of MYO4 or SHE3 leads to delocalization of ASH1 mRNA, resulting in repression of the HOp-ADE2 reporter. In contrast, the khd1Δ mutation had little effect on HO expression (Figure 5A). The frequency of mating-type switching in the khd1Δ mutant was the same as that in the wild-type strain (data not shown). We then examined whether the khd1Δ mutation affected the phenotype associated with a weak myo4-910 mutation, which by itself had little effect on HO expression. The khd1Δ myo4-910 double mutant showed greatly reduced growth on the SC-Ade plate, indicating a reduced level of HO expression in these cells (Figure 5A). This reduced growth of the khd1Δ myo4-910 double mutant on the SC-Ade plate was dependent on the ASH1 gene, because disruption of the ASH1 gene suppressed the growth defect (data not shown). Thus, the khd1Δ deletion enhanced the effect of the myo4 mutation on HO expression, indicating that the KHD1 gene genetically interacts with MYO4. Figure 5.Genetic interactions between KHD1 and SHE. (A) Yeast strains YKEN251 (WT HOp-ADE2-HO 3′ UTR), YKEN252 (myo4-910 HOp-ADE2-HO 3′ UTR), YKEN254 (myo4-910 khd1Δ HOp-ADE2-HO 3′ UTR) and YKEN253 (khd1Δ HOp-ADE2-HO 3′ UTR) were streaked on SC-Ade or SC plates and incubated for 3 days at 30°C. (B) Yeast strains YKEN301 (WT GAL1p-KHD1 HOp-ADE2-HO 3′ UTR), YKEN302 (she3Δ GAL1p-KHD1 HOp-ADE2-HO 3′ UTR), YKEN303 (myo4Δ GAL1p-KHD1 HOp-ADE2-HO 3′ UTR), YKEN304 (ash1Δ HOp-ADE2-HO 3′ UTR), YKEN305 (she3Δ ash1Δ HOp-ADE2-HO 3′ UTR) and YKEN306 (myo4Δ ash1Δ HOp-ADE2-HO 3′ UTR) were streaked on SC-Ade or SG-Ade plates and incubated for 3 days at 30°C. Download figure Download PowerPoint To analyze further the genetic interactions between KHD1 and SHE genes, we examined the effect of KHD1 overexpression on HO expression in myo4Δ and she3Δ mutants. Overexpression of KHD1 from the GAL1 promoter prevented the reduction in HO expression in myo4Δ and she3Δ mutants (Figure 5B). These results suggest a possible genetic interaction between the KHD1 and SHE genes, and imply that Khd1 affects ASH1 mRNA localization at a step different from that of the She proteins. Overexpression of KHD1 inhibits translation of ASH1 mRNA How does KHD1 overexpression suppress the effect of she mutations on HO expression? Since ASH1 negatively regulates the HOp-ADE2 reporter, disruption of the ASH1 gene can suppress a defect in HO expression in she mutants (Figure 5B). This observation raises the possibility that overexpression of KHD1 suppresses the decreased expression of HO observed in she mutations by decreasing Ash1 protein concentrations. To test this possibility, we measured the amounts of myc-tagged Ash1 protein after induction of KHD1 expression from the GAL1 promoter. Western blotting analysis revealed that KHD1 overexpression reduced the concentration of Ash1myc 3.6-fold (Figure 6A). This reduction did not result from toxicity induced by KHD1 overexpression, as the concentration of the unrelated Tub1 protein was not changed (Figure 6A). Overexpression of KHD1 did not affect the concentration of ASH1 mRNA (Figure 6B). These results suggest that KHD1 may be involved in translational control of ASH1 mRNA. Figure 6.Overexpression of KHD1 inhibits translation of ASH1 mRNA. (A) Effect of KHD1 overexpression on Ash1myc protein concentration. Yeast cells were cultured in 2% raffinose medium at 30°C and treated with galactose (2%) to induce KHD1 expression from GAL1p-KHD1. At the times indicated, cells were harvested and western blot analysis was performed to assay the concentration of Ash1myc protein. The concentration of tubulin protein was measured as a quantity control. Strain used: K5552 (Ash1myc) transformed with pK736 (GAL1p-KHD1). (B) Effect of KHD1 overexpression on ASH1 mRNA concentration. ASH1 transcripts were quantitated by northern blotting as described in Materials and methods. rRNA was included as a quantity control. (C) Effect of KHD1 overexpression on ASH1 mRNA localization. Yeast cells were cultured in 2% raffinose medium at 30°C and treated with galactose (2%) for 3 h to induce KHD1 expression from GAL1p-KHD1. ASH1 mRNA was stained by digoxigenin-labeled ASH1 antisense probe (ASH1 mRNA; arrow), and DNA was stained by 4,6-diamino-2-phenylindole (DAPI). Strains used: K5552 (ASH1myc; wild type), YKEN307 (ASH1myc; GAL1p-KHD1). (D) The percentages of cells showing different patterns of ASH1 mRNA localization. Localization was determined by RNA in situ hybridization and classified as follows: anchored: tightly localized ASH1 mRNA at the distal tip; delocalized in the bud: delocalized ASH1 mRNA confined to the bud; delocalized in mother and bud: ASH1 mRNA in both mother cell and bud; neck: ASH1 mRNA at the bud neck. Download figure Download PowerPoint We next examined the effect of KHD1 overexpression on ASH1 mRNA localization. ASH1 mRNA was found to be delocalized in the strain overexpressing KHD1 (Figure 6C and D). In the wild-type strain, 76% of ASH1 mRNA was localized at the distal cortex of the bud. When KHD1 was overexpressed, ASH1 mRNA was localized diffusely within the bud (47%), or mother and bud (17%). These results suggest that the inhibition of ASH1 mRNA translation by KHD1 overexpression might result in a decrease in anchored ASH1 mRNA. Translation of ASH1 mRNA affects its proper localization The observation that KHD1 may regulate the localization of ASH1 mRNA via regulation of ASH1 translation raised the possibility that ASH1 mRNA translation could in turn affect ASH1 mRNA localization. ASH1 mRNA is thought to be translated at the distal tips of daughter buds, with Ash1 protein then transported to the proximal, daughter nuclei. To address whether translation of ASH1 mRNA affects its own localization, we compared localization of wild-type ASH1 mRNA with that of an ASH1 mRNA lacking its initiator ATG codon. Both versions of the ASH1 transcript were placed under the control of the GAL1 promoter to create the constructs GAL1p-ASH1 and GAL1p-ASH1atg−. Western blot analysis confirmed that the mRNA derived from GAL1p-ASH1atg− failed to produce Ash1 protein. RT–PCR analysis showed that this transcript was present at the same concentration as wild-type ASH1 mRNA (Figure 7A). However, in comparison to wild-type ASH1 mRNA, ASH1atg− mRNA was found to be somewhat delocalized in the bud (Figure 7B and C). Whereas 60% of wild-type ASH1 mRNA localized at the distal cortex of the bud, 74% of ASH1atg− mRNA localized diffusely within the bud. These results suggest that translation of ASH1 mRNA has a role in anchoring ASH1 mRNA at the distal cortex of daughter cells. Figure 7.Translation of ASH1 mRNA is required for proper localization of ASH1 mRNA. (A) Expression of Ash1myc protein and ASH1 mRNA. Yeast cells were cultured in 2% raffinose medium at 30°C and treated with galactose (2%) to induce ASH1 expression from GAL1p-ASH1myc and GAL1p-ASH1atg−myc. At the times indicated, cells were harvested and western blot analysis was performed to assay the concentration of Ash1myc protein. Tubulin protein was included as a quantity control. RNAs were also extracted from cell extracts and used as templates for RT–PCR. A 360 bp product was amplified using ASH1-specific primers. One-fifteenth of each reaction was separated on a 2% agarose gel and stained with ethidium bromide. ADH1 mRNA was included as a quantity control. (B) Comparison of ASH1 mRNA localization in TTC356 (GAL1p-ASH1myc; wild-type) and in TTC360 (GAL1p-ASH1atg−myc). Wild-type ASH1 mRNA was localized at the distal cortex of the bud; ASH1atg− mRNA was localized diffusely within the bud. ASH1 mRNA was stained by digoxigenin-labeled ASH1 antisense probe (ASH1 mRNA; arrow), and DNA was stained by DAPI. (C) The percentages of cells showing different pat" @default.
• W2104514904 created "2016-06-24" @default.
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• W2104514904 date "2002-03-01" @default.
• W2104514904 modified "2023-10-16" @default.
• W2104514904 title "The Khd1 protein, which has three KH RNA-binding motifs, is required for proper localization of ASH1 mRNA in yeast" @default.
• W2104514904 cites W105110733 @default.
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