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).
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.
I would like to thank TAs 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
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