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Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation

Iciar L. Ochotorena^1,*,, Dai Hirata^2,*, Kin-ichiro Kominami^1,, Judith Potashkin³, Fikret Sahin⁴, Kelly Wentz-Hunter³, Kathleen L. Gould⁵, Kazuhito Sato², Yasuko Yoshida², Leah Vardy¹ and Takashi Toda^1,¶

¹ Laboratory of Cell Regulation, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
² Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, and `Unit Process and Combined Circuit', PRESTO, JST, Higashi-Hiroshima 739-8526, Japan
³ Department of Cellular and Molecular Pharmacology, Finch University of Health Science, The Chicago Medical School, North Chicago, IL 60064, USA
⁴ Department of Microbiology and Immunology, Finch University of Health Science, The Chicago Medical School, North Chicago, IL 60064, USA
⁵ Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University, Nashville, TN 37232, USA
^* These authors contributed equally to this work
Present address: Nomura Research & Advisory Co. Ltd., Urbannet Otemachi Building 2-2-2, Otemachi, Chiyoda-ku, Tokyo 100-8130, Japan
Present address: Fundacion Inbiomed. Paseo Mikeletegi 61, bajo, 20009 San Sebastian, Gipuzkoa, Spain
^¶ Author for correspondence (e-mail: toda{at}icrf.icnet.uk )

View larger version (36K): [in a new window]   Fig. 1. Defective phenotypes of the wat1 mutant and gene disruption. (A) Diploidisation. Wild-type (top) pop1-364 (middle) or wat1-5235 cells (bottom) were grown in rich medium at 27°C and processed for flow cytometry (FACS). The left panels show the DNA content of individual cells on the x-axis in a logarithmic scale and frequency at the y-axis, while the right panels show forward scattering on the x-axis and a DNA content on the y-axis. (B) Temperature and cold sensitivity. Wild type (left) or wat1-5235 cells (right) were streaked on rich plates and incubated at 19°C, 27°C or 36°C. (C) Tetrad analysis. Two sets of tetrads, derived from heterozygous diploids for the wat1+ gene (I030, Table 1) and grown at 27°C are shown. (D) Diploidisation of the wat1 disruptant. wat1-deleted mutants were streaked on rich medium containing phloxine B and incubated at 27°C for 3 days. White colonies (arrows) show haploid cells, while dark red colonies (arrowheads) are diploid cells (confirmed by FACS).

View larger version (71K): [in a new window]   Fig. 2. Amino acid comparison between Wat1 and homologues from other eukaryotes. Amino acid sequence comparison of Wat1 and homologues from human, rat, fly, budding yeast and plant. Identical amino acid residues are emphasised by blue boxes, and, in particular, invariant amino acid residues that are conserved in all the organisms are shown by dark-blue boxes. WD repeats are underlined.

View larger version (16K): [in a new window]   Fig. 3. Interaction between Wat1 and U2AF-Prp2. (A,B) Protein extracts were prepared from a Wat1-HA (A, lane 2 and B) or a non-tagged control strain (A, lane 1) and immunoprecipitation was performed with anti-HA antibody (A), anti-Prp2 antibody (lane 2 in B) or preimmune serum (lane 1, B), and immunoblotted with anti-Prp2 (A) or anti-HA antibody (B).

View larger version (56K): [in a new window]   Fig. 4. The wat1 mutant shows reduced -tubulin levels. (A) Reduction of -tubulin levels but not other proteins. Protein extracts were prepared from exponentially growing wild-type or wat1-5235 strain, run on SDS-PAGE and immunoblotting was performed with various antibodies shown. (B) Decrease of -tubulin levels upon upwards temperature shift. Wild-type (lanes 1, 4 and 7), wat1-5235 (lanes 2, 5 and 8) or wat1-deleted cells (lanes 3, 6 and 9) were grown at 27°C (lanes 1 to 3) and shifted up to 36°C. After 2 (lanes 4 to 6) and 4 hours (lanes 7 to 9), protein extracts were prepared and immunoblotting was performed with anti--tubulin (top) or anti-Cdc2 antibody (bottom).

View larger version (69K): [in a new window]   Fig. 5. Compromised structure and function of microtubules in the wat1 mutant. (A) Impaired microtubules. Wild-type (left) or wat1-deleted cells were grown at 26°C (middle) and shifted to 36°C for 2 hours (right). Cells were fixed and processed for immunofluorescence microscopy using anti--tubulin antibody. Images from a confocal microscope are shown. Note that wat1-deleted cells are bigger than wild type because they are diploid. (B) Unequal chromosome separation with microtubule defects. wat1-deleted cells grown at 26°C (upper) and 36°C (for 2 hours, lower) were processed for immunofluorescence microscopy as in A and observed under a conventional microscope after staining with DAPI. Anti-tubulin staining (left), DAPI (middle) and merged images (right) are shown. (C) Hypersensitivity to thiabendazole (TBZ). Cells of wild type, wat1-5235, wat1-deleted, TBZ-resistant nda3-ben1 (Yamamoto, 1980) and TBZ-supersensitive atb2 mutants (Adachi et al., 1986) were spotted onto rich plates in the absence (-TBZ, left) or presence of thiabendazole (+TBZ, 20 µg/ml, right) as serial dilutions (106 cells in the left row and then diluted 10-fold in each subsequent spot rightward) and incubated at 26°C for 3 days. Scale bars: 10 µm in A,B.

View larger version (53K): [in a new window]   Fig. 6. mRNA levels are decreased in the wat1 mutant. (A) Northern analysis of tbp1+ transcripts with specific probes for intron and exon sequences. Wild-type (lanes 1 to 3), ts prp2 mutants (lanes 4 to 6) or wat1-deleted cells (lanes 7 to 9) were shifted from 26°C to 36°C, and total RNAs were prepared at 0 (lanes 1, 4 and 7), 2 hours (lanes 2, 5 and 8) and 4 hours (lanes 3, 6 and 9). 20 µg RNA was run in each lane. Northern hybridisation was performed using probes specific for intron (upper) or exon sequence (lower) corresponding to the tbp1+ gene. (B) Steady state transcript levels of various genes. RNA samples from wild-type (lanes 1 and 2) or wat1-deleted cells (lanes 3 to 5) prepared in A were used to examine transcript levels of nda2+, cdc2+, cig2+ and act1+. Equal loading (20 µg) of RNA was confirmed with ethidium bromide staining of the gel (not shown).

View larger version (27K): [in a new window]   Fig. 7. A complex formation of wild-type Wat1, but not mutant protein and immunopurification of the Wat1-containing complex. (A,B) Gel filtration chromatography. Soluble cell extracts were prepared from a wild type-tagged (A, Wat1-HA) or wat1-5235 mutant-tagged strain (B, Wat1-5235-Myc) and loaded onto Superose 6 columns. Each fraction together with total extracts (10 µg, shown as T) was run on an SDS-PAGE and immunoblotting was performed with anti-HA (A) or anti-Myc antibody (B). Positions of size markers (2000 kDa, 669 kDa and 43 kDa) are also shown. (C) Autoradiogram of immunoprecipitated proteins from a Wat1-HA strain with the anti-HA antibody is shown. Cell extracts were prepared from a Wat1-HA (lane 1) or non-tagged wild-type strain (lane2), which was metabolically labelled with Tran[35S]-label. Protein bands that are specifically precipitated with anti-HA antibody are marked by arrows, and the band corresponding to Wat1-HA is also shown (identified with immunoblotting). The positions of molecular weight markers are on the right.

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