Abstract
A temperature-sensitive (ts) mutant of the CHO-K1 cell line, tsTM18, grows at 340C but not at 390C. Smu1 is the gene responsible for ts defects of tsTM18 cells. Previously, we found that the Smu1 ts defect altered the localization (as indicated by enlargement of speckles) of SRSF1 (SF2/ASF) in tsTM18 cells cultured at 390C, suggesting a functional association between Smu1 and SRSF1. Speckles are subnuclear structures that may function as storage/assembly/ modification compartments to supply splicing factors to active transcription sites. The effect of the ts defect of Smu1 on the localization of other factors related to splicing has not been characterized yet. The mechanisms underlying the enlargement of speckles of SRSF1 remain unclear. In the present study, we found that the ts defect of Smu1 affected the nuclear localization of a splicing factor, SRSF2 (SC35), and factors involved in the exon-exon junction complex, Y14 and ALY. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the ts defect of Smu1 affected alternative splicing of endogenous Clk1/ Sty and SRSF2 genes. Mammalian Clk family kinases are shown to phosphorylate serine/arginine (SR) proteins in vitro and SRSF1 in vivo. RT-PCR analysis of Clk1/Sty showed an accumulation of the truncated form lacking kinase activity in tsTM18 cells incubated at 39?C. These data indicate that an accumulation of kinase-negative Clk1/Sty may lead to alteration of the localization of factors related to splicing resulting in the enlargement of speckles.
Highlights
Splice site selection in higher eukaryotes is determined by multiple factors involving cis-acting regulatory elements and their binding proteins
We found that the ts defect of Smu1 affected the nuclear localization of a splicing factor, SRSF2 (SC35), and factors involved in the exon-exon junction complex, Y14 and ALY
We found that the ts defect of Smu1 appeared to alter the localization of SRSF1 in tsTM18 cells cultured at 39 ̊C, suggesting a functional association between Smu1 and SRSF1 [9]
Summary
Splice site selection in higher eukaryotes is determined by multiple factors involving cis-acting regulatory elements and their binding proteins. It is suggested that speckles are dynamic structures and may function as storage/assembly/modification compartments to supply splicing factors to active transcription sites. When transcription or pre-mRNA splicing is inhibited, splicing factors accumulate predominantly in enlarged, rounded speckles [6]. We found that introducetion of a hybrid gene encoding Smu tagged with green fluorescent protein allowed tsTM18 cells to grow at 39 ̊C, resulting in co-localization with SRSF1 [9]. We found that the ts defect of Smu appeared to alter the localization (indicated by enlargement of speckles) of SRSF1 in tsTM18 cells cultured at 39 ̊C, suggesting a functional association between Smu and SRSF1 [9]. Judging from our previous results, Smu may function in the proper localization of SRSF1 at the sites of mRNA processing. We identified the altered localization in a Smu1-dependent manner of proteins involved in mRNA processing, SRSF2, ALY, and Y14
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