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Molecular mechanisms regulating the alternative splicing of the hyaluronan binding protein, CD44

University of British Columbia

CD44 is an extensively glycosylated, widely expressed cell surface protein found on most cells. Multiple protein isoforms of CD44 are generated by the alternative splicing of one or more of 10 contiguous exons into a single site in the processed mRNA, resulting in the inclusion of additional amino acid sequence into the extracellular domain. The expression of certain CD44 isofoms such as CD44R1, which contains additional exons v8, v9 and vlO, has been clinically correlated with an increase in tumour progression and metastatic potential. However, the molecular mechanisms which regulate the alternative splicing of CD44 exons, and thus modulate protein expression, are unknown. A novel in vivo pre-mRNA splicing assay, termed the splice activated gene expression (SAGE) assay, was developed to investigate the pre-mRNA splicing of the CD44R1 variant region exons. This assay consists of a stably expressed in vivo splice construct composed of two or more exons of CD44R1 along with their accompanying intron(s) (v8-v9, v9-vl0 or v8-v9-vl0) placed in frame between the CD44 leader sequence, and a leaderless reporter construct of human liver/bone/kidney alkaline phosphatase (ALP). The expression and enzymatic activity of the ALP is thus dependent upon the prior correct splicing of the CD44 exons. Pre-mRNA splicing is assessed by either enzymatic assay, fluorescence activated cell sorting (FACS) or reverse transcription and polymerase chain reaction (RT PCR). Using this assay system, combined with sequencing of the v8-v9-vl0 genomic regions, alternative 5' and 3' splice sites were identified within the intron (intron 9) which separates exons v9 and vlO. Splicing reactions which utilized these intronic sites, either together or in conjunction with the exonic vl0-3' splice site, resulted in the production of aberrant splice products which retained part of intron 9 including an in frame stop codon. Examination of several leukemic cell lines confirmed the presence and use of these intronic splice sites during native CD44 splicing. Similar intronic splice sites were not found within intron 8. Incubation of K562 cells expressing the SAGE constructs with the serine/threonine kinase inhibitor staurosporine, resulted in an increase in exonic splice site usage at the expense of the intronic splice sites. Thus the activity of several serine/threonine kinases, such as protein kinase C are implicated in the molecular control of CD44 alternative exon splicing.

Thesis/Dissertation

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2009-03-06

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http://hdl.handle.net/2429/5711

Genetics

University of British Columbia

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