Identification and characterization of multiple splice variants of Cdc2-like kinase 4 (Clk4)

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1 Identification and characterization of multiple splice variants of Cdc2-like kinase 4 (Clk4) Vahagn Stepanyan Department of Biological Sciences, Fordham University Abstract: Alternative splicing is an important process that contributes to human proteomic complexity. Protein coding genes undergo alternative splicing, which allows for one gene to code for at least two distinct proteins. Alternative splicing plays an important role in biological processes such gene expression regulation and cell growth (Ghinga et. al., 2008). Deregulation of splicing programs has been linked to inherited and acquired genetic disorders and cancer ( Ghinga et. al., 2008). Incorrect splicing can be due to faulty splicing machinery, which is regulated by essential splicing factors that rely on phosphorylation to carry out their function (Tazi et. al., 2009). The phosphorylation of these essential splicing factors is carried out by Cdc2-like kinases 1,2,3, and 4 (Clk1, 2, 3, and 4), which themselves are subject to alternative splicing. Using RT-PCR we identified and characterized previously unreported splice variant of Clk4 in AG10587A (lymphocyte) and HepG2 (hepatocellular carcinoma) cell lines. Introduction: It has been shown that a small number of genes contribute to human proteomic complexity. This is achieved through process of alternative splicing pre-mrna, which allows for one gene encodes at least two structurally and functionally distinct protein isoforms (Hagiwara, 2005). A gene is transcribed into primary RNA (pre-mrna) that

2 contains intronic and exonic regions, mrna splicing machinery removes the intronic regions and exons are joined to from mature mrna. Depending on what the cell decides, different number of exons of the same pre-mrna can be spliced out and joined together into a mature mrna and thereby may code for different proteins isoforms (Tazi et. al., 2009). It is estimated that 35-60% of human genes encode two or more alternatively spliced isoforms (Eisenreich et. al., 2009). Regulation of splice sites allows for the control of gene expression and for generation of proteomic diversity, which play an important role in many biological processes such as embryonic development, cell growth, and apoptosis (Eisenreich et. al., 2009). Splicing defects in specific genes have been linked to genetics disorders such as spinal muscular atrophy, myotonic dystrophy, β-thalassemia and many others. Cancer development has also been linked to deregulation of alternative splicing. ( Ghinga et. al., 2008) This deregulation promoted by the activity of splicing regulators can lead expression of tumor-specific variant proteins (Ghinga et. al., 2008). A ribonucleoprotein complex known as the spliceosome carries out splicing (Hagiwara, 2005). Spliceosome activity is assisted by essential splicing factors such as serine/arginine (SR) rich proteins, which promote splice site recognition and commit spliceosome to pre-mrna splicing (Colwill et. al., 1996). SR proteins family have at least one RNA recognition motif on their N terminus and have an Arginine and Serine (RS) rich domain on their C terminus (Colwill et. al., 1996). All SR proteins are phosphorylated at the RS domain that facilitates their proteinprotein interaction, prevents SR proteins from randomly binding to mrna, and are thought to promote spliceosome assembly. Once spliceosome is assembled SR

3 proteins are dephosphorylated ( Eisenreich et. al., 2009). The phosphorylation of SR proteins, particularly SF2/ASF (Alternative splicing factor SF2/ Alternative splicing factor) is carried out by a family of Cdc2-like kinases 1, 2, 3, and 4 (Clk1, 2, 3, and 4) (Hagiwara, 2005). Clk s phosphorylate Serine, Threonine, and Tyrosine residues. Pre-mRNA of Clk is subject to constitutive and alternative splicing generating. (Nayler et. al., 1997). Clk1 is an important and well-studied alternative splicing regulator and shares 69% amino acid identity with Clk4. There are 4 isoforms of Clk1 predicted on NCBI. Since there is only one form of Clk4 reported, the purpose of this study is to identify and analyze potential Clk4 splice variants. Methods: RNA was isolated from AG10587A (Lymphocyte) and HepG2 (Hepatocellular carcinoma) cell lines using RNeasy Plus Mini Kit (QIAGEN). RT-PCR was performed using One-Step RT-PCR Kit. Primers were designed to span exons 2 and 6 of Clk4 and in exons 2 and 5 of Clk1 were used for Lymphocyte and HepG2 cell lines. The RT-PCR products were run on 1% agarose gel at 160V. The bands were visualized using UV light. Desired bands were extracted using a QIAquick Gel extraction kit and sent for DNA sequencing.

4 Results: A B Fig. 1 Fig. 1 Visualization of RT-PCR products of Clk1 and Clk4 in lymphocyte and HepG2 cell lines RT-PCR was performed using primer in exons 2 and 5 for Clk1 and in exons 2 and 6 for Clk4. Fig. 1 A shows the visualization of RT-PCR product performed on RNA isolated from AG10587A (Lymphocyte), which shows alternative splicing of both Clk1 and Clk4, and that band 1 of Clk1 and Clk4 and band 2 of Clk1 and Clk4 are of the same molecular size. Fig. 1 B shows visualization of RT-PCR product performed on RNA isolated from HepG2 (Hepatocellular carcinoma) cells to see whether or not Clk4 alternative splicing is unique to lymphocytes. The results showed that Clk1 and Clk4 spliced, and band 1 of Clk1 and Clk4 and band 2 of Clk1 and Clk4 are of the same molecular size.

5 Bands 1 and 2 of Clk1 and Clk4 of both cell lines were extracted and sent for DNA sequencing. The analysis of the DNA sequences of the bands showed that band 1 in both AG10587A (Lymphocyte) and HepG2 cell line contained all the exons the primers were designed to span. However, sequence analysis of band 2 of AG10587A (Lymphocyte) and HepG2 cell line showed that 91 base pair sequence had been spliced out. This sequence corresponds to exon 4. To see what effects if any the splicing out of exon 4 had on amino acid sequence the amino acids coded for by exons 1 to 5 in two alternatively spliced Clk1 transcripts were aligned in Figure 2. Fig. 2 Alignment of amino acids coded for by Exons 1 to 5 in two alternatively spliced Clk1 transcripts The top sequence in Fig. 2 is exon 4 containing and the bottom sequence is exon 4 lacking. The highlighted amino acids of both sequences are the same. These sequences are coded by exons 1 to 3 which are unaffected by splicing. However, the splicing out 91 base pair exon 4 introduces an early stop codon. The 91 st base of exon 4 and first two bases of exon 5 form a codon. Splicing out of exon 4 shifts the coding frame, which results in premature stop codon. The amino acid tyrosine (Y) that is highlighted with red marks the beginning of kinase domain, which the exon 4 lacking sequence does not code for.

6 To see what effects if any the splicing out of exon 4 had on amino acid sequence the amino acids coded for by exons 1 to 6 in two alternatively spliced Clk4 transcripts were aligned in Figure 3. Fig. 3 Alignment of amino acids coded for by Exons 1 to 6 in two alternatively spliced Clk4 transcripts The top sequence in Fig. 3 is exon 4 containing and the bottom sequence is exon 4 lacking. The highlighted amino acids of both sequences are the same. Similar to pervious result, the highlighted amino acid sequence are identical in both exon 4 containing and lacking sequences. However, the splicing out 91 base pair exon 4 introduces an early stop codon. The 91 st base of exon 4 and first two bases of exon 5 form a codon. However, splicing out of exon 4 shifts the coding frame, which results in premature stop codon and a truncated protein. The amino acid tyrosine (Y) that is highlighted with red marks the beginning of kinase domain, which the exon 4 lacking sequence does not code for. When the exon 4 of Clk1 and Clk4 are aligned apart from 6 bases the exon 4 sequences of Clk1 and Clk4 are identical, suggesting that there might be common regulation sequence that is playing a role in the alternative splicing of this exon.

7 Discussion: This project showed that Clk1 spliced into variants as reported on NCBI. We showed that first band of Clk1 was full length variant, and band two was exon 4 lacking variant which resulted in shift of reading frame and introduced early stop codon. However, we also found a previously unreported splice variant of Clk4 in both lymphocyte and HepG2 cell lines. DNA sequencing of the RT-PCR products showed that the band with heavier molecular weight corresponded to full length reported gene. However, band two of Clk4, with smaller molecular weight, had exon 4 spliced out, which resulted in shift of reading frame and introduction of early stop codon. This alternatively spliced variant does not code for the kinase domain due to the premature stop codon, and hence cannot phosphorylate essential splicing factors in these cell lines. NCBI needs to be updated to include this splice variant of Clk4. Acknowledgements: I would like to thank TA s Anthony Evans and Faaria Fasih-Ahmad for always being in class to assist with the projects, for provide us with necessary materials, and without whose tireless help this project would not have been possible. I would also like to thank Dr. Berish Rubin for his help and support to make this project possible.

8 References: Colwill K. et al.. SRPK1 and Clk/Sty protein kinases show distinct substrate specificities for serine/arginine-rich splicing factors. The Journal Of Biological Chemistry. 1996;271: Eisenreich A. et al.. Cdc2-like kinases and DNA topoisomerase I regulate alternative splicing of tissue factor in human endothelial cells. Circulation Research. 2009;104: Ghigna C., Valacca C., Biamonti G. Alternative splicing and tumor progression. Current Genomics. 2008;9: Hagiwara M. Alternative splicing: a new drug target of the post-genome era. Biochimica et Biophysica Acta. 2005;1754: Nayler O, Stamm S, Ullrich A. Characterization and comparison of four serine- and arginine-rich (SR) protein kinases. Biochem J. 1997;326: Tazi J, Bakkour N, Stamm S. Alternative splicing and disease. Biochimica et Biophysica Acta. 2009;1792(1):14 26.