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

Vahagn Stepanyan


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 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).
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).
Cdc2-like kinases 1,2,3, and 4 (Clk1, 2, 3, and 4) phosphorylate essential splicing factors. 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.


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.


Fig. 1 A and B show the visualization of RT-PCR product performed on RNA isolated from AG10587A (Lymphocyte) and HepG2 cell lines, 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.
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 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. Splicing out of exon 4 shifts the coding frame, which results in premature stop codon and a truncated protein.


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. NCBI needs to be updated to include this splice variant of Clk4.


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;271:24569–24575.

Eisenreich A. et al. . Cdc2-like kinases and DNA topoisomerase I regulate alternative
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Tazi J, Bakkour N, Stamm S. Alternative splicing and disease. Biochimica et Biophysica Acta. 2009;1792(1):14–26.


Figure 1-Visualization of RT-PCR products of Clk1 and Clk4 in lymphocyte and HepG2 cell lines

Figure 2-Alignment of amino acids coded for by Exons 1 spliced Clk1 transcripts to 5 in two alternatively

Figure 3-Alignment of amino acids coded for by Exons 1 to 6 in two alternatively spliced Clk4 transcript

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.

Full Paper


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

This document was last modified 05/16/2018.
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