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.
Sugaya
Kimihiko
K
Radiation Effect Mechanisms Research Group, National Institute of Radiological Sciences, 4-9-1, Anagawa, Inage-ku, Chiba 263-8555, Japan. k_sugaya@nirs.go.jp
Hongo
Etsuko
E
Ishihara
Yoshie
Y
Tsuji
Hideo
H
D017398:Alternative Splicing; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000096:biosynthesis; D019143:Evolution, Molecular; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D050505:Mutant Proteins / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
D019812:Heparan Sulfate Proteoglycans; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C071927:perlecan; D004247:DNA