Pre-mRNA splicing by the essential Drosophila protein B52: tissue and target specificity.

Many genes are interrupted by introns that are removed in the process of making a functional mRNA and, ultimately, a protein product. This splicing process takes place in a megadalton RNA-protein complex termed the spliceosome. The spliceosome consists of many components, including five small nuclear ribonucleoprotein particles (snRNPs) each consisting of a small nuclear RNA and a complement of proteins (17). Additionally, 100 or more non-snRNP proteins are present in the spliceosome (23). One group of non-snRNP proteins that has been studied intensively since their discovery is the SR protein family (8). SR proteins have a similar protein structure, with at least one RNA recognition motif (RRM) at the N terminus and a region rich in arginine-serine dipeptides (RS domain) at the C terminus. The RRM consists of 80 to 90 amino acids; is present in many RNA-binding proteins, including members of the SR protein family; and confers RNA binding specificity (13, 20). The RS domain has been postulated to mediate protein-protein interactions between SR family members (15, 39, 40). The SR protein family includes proteins with sizes of 20, 30, 40, 55, and 75 kDa, and the high degree of conservation from Drosophila to humans indicates the importance of these proteins (8). This family of SR proteins, purified by a two-step salt precipitation procedure, has a well-defined biochemical activity, such that each member can complement a splicing-deficient cytoplasmic (S100) extract, indicative of a functional redundancy among SR proteins. In Drosophila melanogaster, only two SR proteins, RBP1 and B52, have been cloned and characterized to date. RBP1, homologous to human SRp20, is expressed in all tissues throughout development and colocalizes with RNA polymerase II on larval salivary gland polytene chromosomes. RBP1 also tested positive for splicing activity in a cell-free human splicing system (14). B52, the Drosophila homolog of human SRp55 is present during all stages of development and was initially characterized because of its localization to heat shock puffs on polytene chromosomes. B52 brackets RNA polymerase at the largest heat shock puffs and stains within developmental puffs (4, 18). The proper level of expression of B52 is critical for development of the animal. Overexpression of B52 in various tissues results in developmental arrest in almost all cases (18). The generation of a B52-null mutant showed that B52 expression was essential, given that these animals do not develop past the second-instar larval stage. Surprisingly, several genes analyzed thus far show proper splicing patterns in arrested larvae of the B52-null mutant (22, 24). Thus, a global RNA processing defect may not explain the lethality of the B52 deletion mutation. In order to understand the cause of the lethality of the null mutation, we sought to characterize more thoroughly the splicing activity of B52 in vivo and in vitro. Here, we show that B52 is essential for splicing an ftz pre-mRNA in Drosophila extracts in which B52 is the predominant SR protein. In vivo, other SR proteins appear to complement the B52 deficiency in most tissues. However, by analyzing RNA isolated from the brain, a tissue in which B52 is the major SR protein expressed, we showed that the production of ftz mRNA is dependent on B52. These results support the hypothesis that the lethality seen in the B52-null mutant is a consequence of splicing defects in tissues that lack sufficient concentrations of other SR protein family members.