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

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The conserved role of Smu1 in splicing is characterized in its mammalian temperature-sensitive mutant

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

^* Author for correspondence (e-mail: k_sugaya{at}nirs.go.jp)

Accepted 3 October 2006

Temperature-sensitive CHO-K1 mutant cell line tsTM18 exhibits chromosomal instability and cell-cycle arrest at S and G2 phases with decreased DNA synthesis at the nonpermissive temperature, 39°C. We previously identified an amino acid substitution in Smu1 that underlies the temperature-sensitive phenotypes of tsTM18 cells. In the present study, we confirmed that Smu1 is associated with the temperature-sensitive defect of tsTM18 by RNA interference. We also found an early temperature effect in DNA synthesis. Because genetic studies of nematodes revealed that smu-1 is involved in splicing of the unc52/perlecan pre-mRNA, we analysed the perlecan transcript in tsTM18 cells by reverse transcription-polymerase chain reaction (RT-PCR). The perlecan PCR product amplified from RNA of tsTM18 cells cultured at 39°C appeared to be a mixture of variants. Sequence analysis identified at least six variants that result from alternative splicing and intron retention. Comparison of the results of perlecan RT-PCR analysis with those of analysis of four other genes suggested that the splicing defect in the perlecan gene is unique and that it is conserved through evolution.

Key words: Alternative splicing, DNA synthesis, Intron retention, RNA interference, Temperature-sensitive mutation

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