Regulation of Human Telomerase Alternative Splicing
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Telomerase adds TTAGGG repeats onto chromosome ends (telomeres). Since telomerase is expressed in ~90% of all human cancer cells while being absent in most somatic tissues, telomerase is an almost universal cancer therapeutic target. Yet, the clinical application of an effective telomerase inhibitor is still lacking. The pre-mRNA of the catalytic subunit of human telomerase (hTERT) may be alternatively spliced into 22 different isoforms. Only a small fraction of hTERT transcripts are spliced into the full length isoform, the form capable of being translated into function hTERT with reverse transcriptase activity. If telomerase activity is partially regulated at the level of RNA splicing, then telomerase activity may be modulated to increase the amount of nonfunctional transcripts and decrease the amount of full-length hTERT and this would be a novel anti-cancer therapeutic approach. A hTERT minigene was created to understand the cis-regulatory elements governing hTERT splicing. A 1.1kb region of 38 bp repeats (block 6) ~2 kb from the exon/intron junctions is essential for the exclusion of exons 7 and 8. Block 6 repeats suggested that RNA:RNA pairing may regulate splicing of hTERT. Mutations within the repeat sequence that abolish exon skipping were corrected by compensatory mutations in the pre-mRNA. To identify trans-acting regulators of hTERT alternative splicing, the minigene was modified into a dual-luciferase reporter for a selected 528 RNA-binding proteins/splicing factors siRNA screen. Our initial validation focused on 45 factors with enzymatic activity and resulted in CDC-Like Kinase 1 (CLK-1) as a potential hTERT splicing modulator. Knock-down of CLK-1 altered hTERT splicing, resulting in reduction in telomerase activity and telomere shortening. CLK-1 belongs to the Clk family of dual-specificity nuclear kinases that can auto-phosphorylate SR proteins. TG003, a chemical CLK-1/4 inhibitor, results in less full length hTERT splicing and a decrease in cancer cell telomerase activity. This approach provides a platform to identify additional hTERT splicing regulators that may be suitable drug targets to increase or decrease telomerase activity. Altogether, these results demonstrate the potential of manipulating hTERT splicing as a target for both chemotherapy and regenerative medicine.