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First published online 14 November 2006
doi: 10.1242/jcs.03288

Journal of Cell Science 119, 4944-4951 (2006)
Published by The Company of Biologists 2006
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The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant

Kimihiko Sugaya*, Etsuko Hongo, Yoshie Ishihara and Hideo Tsuji

Radiation Effect Mechanisms Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, Japan


Figure 1
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  		Fig. 1. Effect of expression of siRNA against SMU1/Smu1 on colony formation. (A) Diagram of the structures of mammalian Smu1. Black boxes, WD-repeat motifs. Locations of the sequence corresponding to siRNAs are indicated as S1, S2 and S3 above the diagram. An amino acid substitution found in the last WD-repeat of Smu1 of tsTM18 is indicated as G498R. (B) A photograph of colonies of 18H-1c30 cells after transfection of plasmid DNAs encoding siRNAs. Colonies on dishes after 12 days of incubation at 34°C or 39°C with 5 µg ml-1 puromycin were stained with methylene blue. Cells survived at 34°C after transfection of S2 or S3; however, few cells grew at 39°C or at 34°C after transfection of S1. Most cells formed colonies after transfection of an EGFP plasmid (control). (C) Colony formation at 39°C relative to that at 34°C by 18H-1c30 (upper) and 18K-1c1 (lower) cells. The mean and s.d. of six independent experiments are indicated as a solid bar and line, respectively. P values were calculated by Student's t-test. Significant differences in colony formation were found between EGFP and S2 and S3.

 

Figure 2
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  		Fig. 2. Effects of incubation for 4 hours at 39°C on DNA synthesis. (A,B) DNA synthesis activity of CHO-K1 and tsTM18 cells were measured by [3H]thymidine incorporation as described in the Materials and methods. The average and standard deviation of at least three independent experiments are indicated. Incorporation rate at 39°C relative to that at 34°C of replication was 1.34 for CHO-K1 and 0.77 for tsTM18 cells, respectively. The temperature shift appears to reduce DNA synthesis in tsTM18 cells. (C-F) Images of replication were obtained as described in the Materials and methods. Single equatorial sections were collected with a confocal microscope. Scale bar, 10 µm.

 

Figure 3
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  		Fig. 3. Schematic diagram of the structures of the perlecan gene and its splice variants in a hamster smu1 mutant cell line. (A) Schematic structure of the C. elegans, mouse and human homologs of perlecan. The five major domains are indicated by four boxes and ellipses with Roman numerals. The alternatively spliced region of the C. elegans counterpart of perlecan, unc-52, in smu-1 mutant is indicated above the diagram. Exons (15-19) that correspond to Ig-like repeats are represented by boxes with the introns indicated by horizontal lines. The number of Ig repeats in domain IV varies between species. Mouse perlecan has 14 repeats, whereas human perlecan has 21 repeats resulting from the probable insertion of seven Ig-like repeats. (B) RT-PCR analysis of splice variants of the hamster perlecan transcripts. RT-PCR analysis for CHOK1, tsTM4 and tsTM18 cells was carried out as described in the Materials and Methods. A 1661-bp product is present in RNAs from CHO-K1 and tsTM4 cells. Although 1661-bp product was also amplified from RNA isolated from tsTM18 cells incubated at 34°C, RT-PCR products from tsTM18 cells cultured at 39°C were a mixture of several size variants. Similar results were observed in two additional experiments (Fig. 4B and Fig. 5). (C) Positions of primers and structures of splice variants. Lines indicate exon sequences, dashed lines indicate sequences that are spliced out, and vertical lines indicate exon/intron junctions. Retained intron sequences are indicated by a triangle under the transcript.

 

Figure 4
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  		Fig. 4. Retention of intron sequences in the perlecan transcript in hamster tsTM18 cells. Total RNA and genomic DNA of CHO-K1 and tsTM18 cells were treated with or without DNase and then analysed with primer pair f6/r2 by RT-PCR for RNA or by PCR for DNA. (A) After treatment with DNase, a 97-bp product appears in RNA fraction, and a 300-bp product is present in RNA from tsTM18 cells cultured at 39°C. No PCR products were obtained with DNA as template. The closed triangle indicates a transcript with retained intron sequences, and the open triangle indicates the transcript predicted from the sequence. (B) Result of RT-PCR with primer pair f2/r2. Lane numbers correspond to those in panel A. Splice variants are seen in the amplification products of tsTM18 cells incubated at 39°C (lane 5). (C) Results of RT-PCR and PCR without DNase pretreatment. Triangles indicate the product predicted from the sequence. A 300-bp PCR product was amplified from DNA.

 

Figure 5
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  		Fig. 5. Accumulation of splice variants of perlecan in tsTM18 cells during culture at 39°C. Incubation times at 39°C are indicated above the gel. A 1661-bp RT-PCR product was predicted on the basis of the sequence. This product is present in CHO-K1 cells and in tsTM18 cells incubated at 34°C. However, during culture of tsTM18 at 39°C, the amount of the 1661-bp product decreases gradually, and a number of different splice variants appear.

 

Figure 6
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  		Fig. 6. RT-PCR analysis of the Aptx, Dnaja1, Smu1 and Tnc transcripts in CHO-K1 and tsTM18 cells. Amplified regions are indicated by black boxes above the photograph. The coding sequences of Aptx, Dnaja1 and Smu1 were amplified. The FN III-type repeat 5-6 region of Tnc, which is alternatively spliced in mammalian development and in cancer progression (Joester and Faissner, 1999Go), was analysed. There were no changes in expression of Dnaja1, Smu1 and Tnc in CHO-K1 and tsTM18 cells incubated at 39°C. There were at least three size variants of Aptx, and the shortest one appeared in both CHO-K1 and tsTM18 cells during culture at 39°C.

 

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