Functional study of SR splicing factors in a cellular genetic system

Pre-mRNA splicing is a critical and highly regulated process in eukaryotic gene expression. Splicing reaction is conducted by hundreds of protein factors, which form a structure called spliceosome. The SR protein family is a group of regulatory splicing factors whose functions have been well-studied in biochemical systems. Typical SR proteins contain one or two RNA recognition motifs at the N-terminus, and serine-arginine repeats at the C-terminus. SR proteins also play important roles in other steps of mRNA metabolism in the cell. Despite extensive biochemical characterization, the mechanism of SR protein action in the cellular context remains largely elusive. Inactivation of SR proteins by gene targeting indicates that the SR proteins are required for animal development and cell viability. However, the mechanism behind is far from clear. To study how SR proteins act in mammalian cells, we constructed two mouse embryo fibroblast (MEF) cell lines in which the endogenous genes for the SR protein ASF/SF2 and SC35 were each deleted and complemented by prospective exogenous genes expressed from a tetracycline-controlled promoter. Addition of tetracycline/ doxycycline in cell culture efficiently turns off the expression of these SR proteins. The inducible cell lines have been used as a genetic model in functional studies. Several interesting findings observed in these genetic systems form the basis of this dissertation: 1) In mammals, both SR proteins are uniquely required for survival of proliferating cells but dispensable in non-dividing cells. 2) RNA binding motif is required, while RS domain is disposable, for ASF/SF2 function in vivo. 3) Recycling SR proteins in cells is controlled by a sorting mechanism regulated by the phosphorylation state of RS domain. 4) SR proteins are critical for transcriptional elongation in mammalian cells. 5) The elongation defects trigger extensive double- stranded DNA breaks, leading to ATM-mediated activation of p53, inducing p21 expression, and cell cycle arrest. Our research has shed light on a number of fundamental questions regarding the function of SR proteins in regulated mRNA processing, maintenance of genomic stability and cell proliferation in vertebrates. Moreover, our work has established an experimental platform for future exploration of the cellular functions of SR protein