Alternative pre-mRNA splicing is a mechanism of regulation of gene expression that affects at least 60% of human genes. Inaddition, at least 15% of genetic defects are associated with aberrant pre-mRNA splicing, an incidence that reaches 40% formulations that inactive genes encoding for example the tumour suppressor BRCA1 or the gene responsible for neurofibromatosis. The molecular mechanisms controlling this process during cell differentiation and development, andtheir alterations in pthological situations are not well understood. Recent work in the host lab has identified a novel mechanism of splicing regulation whereby the regulatory protein Sex-lethal (SXL) inhibits splicing of a specific intone within its own pre-mRNA at the very last step of the splicing reaction (axon legation) (Lillian et al, 2002). This is in contrast with other mechanisms described previously, which target early events in splice site (sis) recognition. The work also revealed a critical role for the splicing factor SPF45 in selecting particular 3\'sis and in allowing regulation by SXL. SPF45 is the first known splicing factor that recognizes directly the 3\' sag and at the same time activates it for catalysis. Interestingly, SPF45 was also found to be involved in the activation of cryptic 3\' sis located upstream from the natural 3\'sis in the ÿ-globing gene. Activation of the cryptic site, which breaks the open reading frame and therefore prevents the mRNA from encoding functional ÿ-globing protein, is caused by a G to A mutation responsible for one form of ÿ-thalassemia (ÿ110). We propose a structural/functional analysis of SPF45 to understand its activities as
(a) second step splicing factor,
(b) selector of 3\' sis AG for catalysis,
(c) mediator of SXL auto regulation and
(d) activator of the cryptic 3\'sis responsible forÿ110-thalassemia. We also intend to use microarray designed to study alternative splicing and RNA interference technology to identify new regulatory genes.
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