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

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 degrees 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 degrees 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.