Alternative pre-mRNA splicing: factors involved in splice site selection.

The protein-coding regions of most vertebrate genes are split, giving rise to primary transcripts containing introns and exons; the latter must be spliced in the nucleus to form the mature mRNA, which then moves to the cytoplasm for translation. Pre-mRNA splicing, the process of intron removal and exon joining, represents an important function within the nucleus, and elucidation of the mechanisms of splicing has challenged researchers since the discovery of segmented genes. A surprising discovery was that many genes have the capacity to encode multiple protein products as a consequence of alternative splicing events. Thus, a great deal of interest centers around characterizing the mechanisms whereby multiple mRNAs can derive from a common pre-mRNA, especially those events which exhibit celland/or tissue-specific regulation. Important advances have been made recently, with the identification of trans-acting factors that can modulate splice site choice in vitro. However, much of this work stems directly from the tremendous progress that is being made in the characterization of the factors involved in constitutive splicing. The ability of whole cell and nuclear extracts to perform intron removal and exon ligation events has led to the rapid development of a detailed outline of the steps involved in mRNA splicing (reviewed by Green, 1991; Krainer and Maniatis, 1988; Sharp, 1987). Each intron is removed in a twostep process (Fig. 1). First, cleavage at the boundary between the intron and the exon upstream (the 5′ splice site or splice donor) occurs, with concomitant joining of the 5′ phosphate at the 5′ splice site to a 2′ hydroxyl of a residue within the intron, forming a branched nucleotide. This lariat intermediate undergoes cleavage at the junction between the intron and the downstream exon (the 3′ splice site or splice acceptor), with the coordinated ligation of the two free exons to form mature mRNA and the liberation of the lariat intron. In cases of alternative splicing, exons may have multiple 5′ or 3′ splice sites, may be entirely skipped, and introns may be retained; some examples are diagrammed in Fig. 2 (see also McKeown, 1992; Smith et al., 1989).