Abstract
A temperature-sensitive (ts) CHO-K1 mutant cell line, tsTM18, exhibits chromosomal instability with decreased DNA synthesis at the nonpermissive temperature, 39oC. An amino acid substitution in Smu1 underlying the ts phenotypes of tsTM18 cells was identified previously. We also found a ts defect in splicing of the unc52/perlecan gene. In the present study, we have generated cell lines expressing Smu1 tagged with green fluorescent protein (GFP) to study the dynamics of Smu1 in living cells. The hybrids complement deficiencies in tsTM18 cells and allow them to grow normally at 39°C. GFP-tagged Smu1 is found in speckles in many discrete nucleoplasmic sites, and most of these also contained SF2/ASF. SF2/ASF is a member of the serine/arginine (SR)-rich splicing group of factors that are necessary for spliceosome assembly and can influence alternative splicing. SF2/ASF is also involved in the integrity of genome maintenance. In tsTM18 cells cultured at 39°C, the Smu1 ts defect appears to alter SF2/ASF localization, suggesting a physiological association of Smu1 with SF2/ASF. The significant decrease of Smu1 may lead the enlargement of speckles of SF2/ASF. These data show the importance of Smu1 as a regulator of splicing and genome maintenance.
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