Splicing of messenger RNA precursors is a critical step in the long chain of events required for the expression of most eukaryotic genes. The majority of these genes are transcribed as a large precursor mRNA (pre-mRNA) in which the coding regions are interrupted by intervening sequences (introns). In higher eukaryotes, nuclear pre-mRNAs typically contain multiple introns (in some cases greater than 50) with variable lengths extending up to 200,000 nucleotides. The conversion of pre-mRNA to functional mRNA requires the precise excision of these introns and the subsequent ligation of the coding sequences (exons), a process called splicing. Even the smallest of errors in the splicing reaction can have deleterious effects (for example, shifting the reading frame of the mRNA) that result in the production of altered, nonfunctional protein. The splicing of some pre-mRNAs is regulated during development or in a tissue-specific manner, such that alternatively spliced mRNAs, which are either nonfunctional or code for different protein isoforms, are generated from the same pre-mRNA. Alternative splicing thus represents an additional level of genetic regulation that enhances the genetic capacity of eukaryotes.
Much has been learned about the mechanism of nuclear pre-mRNA splicing through biochemical studies in both mammalian and yeast systems, as well as via genetic approaches in yeast. In particular, the development of an in vitro splicing assay has played a key role in identifying components involved in the splicing reaction and elucidating the splicing pathway. These studies have demonstrated that the basic chemical mechanism of splicing is conserved between higher and lower eukaryotes. The removal of an intron and subsequent ligation of the flanking exons is a two-step process that involves two temporally distinct transesterification (ie, phosphate transfer) reactions (see Fig. 1). The phosphodiester bonds of the pre-mRNA that are to be cleaved and then ligated are precisely defined by consensus sequences located around the 5′ and 3′ splice sites. The first step of splicing is initiated by a nucleophilic attack on the phosphate at the 5′ splice site by the 2 ‘ -OH group of an adenosine (designated the branch point), which is normally located 18 to 40 nucleotides upstream of the 3 ‘ splice site. This results in cleavage of the 3′ ,5′ phosphodiester bond at the 5′ splice site and the concomittant formation of an unusual 2 ‘ ,5′ phosphodiester bond between the first nucleotide of the intron and the branch site adenosine. This reaction produces two splicing intermediates, the 5 exon and a circular tailed molecule (ie, a so-called lariat) containing the intron and 3′ exon. In the second step, the 3′ OH of the 5′ exon, which is released by the first transesterification reaction, carries out a nucleophilic attack on the phosphate at the 3 splice site. This results in excision of the intron in the form of a lariat and the simultaneous ligation of both exons via a 3′ ,5′ phosphodiester bond. Despite the fact that pre-mRNA splicing requires the hydrolysis of ATP, the phosphates present in the products of the splicing reaction (ie, the spliced mRNA and excised intron) are derived from the pre-mRNA substrate and not from ATP (1-3). Splicing thus essentially involves the exchange of one pre-mRNA substituent for another on the phosphodiester bond at the 3′ splice site.
Figure 1. Schematic representation of the two-step splicing pathway of nuclear pre-mRNA introns. Boxes and solid lines represent exon and intron sequences, respectively. The consensus sequences found at the mammalian 5 and 3 splice sites and branch site of U2-dependent introns are indicated, where N = any base, Y = pyrimidine, and R = purine. The branch site adenosine is marked with an asterisk, and the polypyrimidine tract is indicated by Yn. The nucleophilic attacks on the splice sites by the 2 ‘ OH of the branch site adenosine (step 1) and of the 3′ OH of the cleaved 5′ exon (step 2) are depicted by dashed arrows. The phosphate groups at the 5′ and 3′ splice sites, which are conserved in the splicing products, are also indicated.
Similar reaction intermediates and products are also observed during the splicing of group II introns, which are found in pre-mRNA molecules synthesized in plant and fungal organelles (4) (see Self-Splicing Introns). This has led to the hypothesis that group II and nuclear pre-mRNA introns may be evolutionarily related and that both types of splicing may involve similar catalytic mechanisms. Group II introns, in contrast to nuclear pre-mRNA introns, can be spliced autocatalytically in vitro in the absence of proteins or other factors. The self-splicing nature of these introns lies in their ability to fold into an elaborate, highly conserved intramolecular structure that favorably aligns the 5 and 3 splice sites and the branch site for the two cleavage/ligation reactions of splicing. Nuclear pre- mRNA introns, on the other hand, possess only short conserved cis-acting sequence elements that are confined to the 5′ and 3′ splice sites, the polypyrimidine tract (present only in higher eukaryotes), and the branch site (see Splice Sites). The folding of nuclear pre-mRNA introns into a catalytically favorable conformation thus requires the presence of a large number of trans-acting factors. These include the small nuclear RNP-(snRNPs), evolutionarily highly conserved ribonucleoprotein (RNA-protein) complexes, and non-snRNP proteins. These factors interact stepwise with the pre-mRNA to form the spliceosome, a large ribonucleoprotein complex that catalyzes both steps of splicing. Two distinct spliceosomes have to date been identified. The major or U2-dependent spliceosome is found in all eukaryotes and is responsible for the excision of so-called U2-dependent introns, which comprise the vast majority of nuclear pre-mRNA introns. The recently identified minor or U12-dependent spliceosome catalyzes the removal of the less abundant U12-dependent introns, which at present have been identified in only a subset of eukaryotes (5-7).
Despite recent advances in the splicing field, our understanding of the complex process of nuclear pre-mRNA splicing is far from complete. Currently little is known about the three-dimensional structure of the spliceosome and the nature of the spliceosomal active sites (i.e., whether the catalyst is RNA and/or protein) remains a matter of intense debate and investigation. One of two structurally distinct active sites appears to be responsible for each catalytic step of splicing (8, 9). During spliceosome assembly, the RNA components of the spliceosomal snRNPs (ie, the UsnRNAs) base-pair in a dynamic fashion with the short conserved regions of the pre-mRNA around the 5′ and 3′ splice sites, as well as with one another. This leads to the formation of a dynamic network of RNA-RNA interactions (see Spliceosome) that apparently provides the structural framework necessary for the catalysis of pre-mRNA splicing (reviewed in Refs. 10 and 11). In fact, structural elements that closely resemble highly structured functional domains characteristic of self-splicing group II introns have been identified in the spliceosomal RNA network, supporting the long-standing hypothesis that nuclear pre-mRNA splicing is largely, if not exclusively, catalyzed by RNA (12). In addition to the snRNAs, however, proteins also play important roles in pre-mRNA splicing, particularly during the assembly of the spliceosome. Over 70 spliceosomal proteins have been identified thus far, and much effort is still currently invested in their structural and functional characterization. Proteins are involved in a number of protein-protein interactions and protein-RNA interactions that are required for the formation of a catalytically active spliceosome (see Spliceosome). In addition, several spliceosomal proteins possessing enzymatic activity (eg, RNA unwindase or protein isomerase activity), which are thought to catalyze conformational changes in the spliceosome during the splicing process, have also been identified (reviewed in Refs. 13 and 14). More recently, proteins have even been proposed to contribute directly to the spliceosome’s active sites. At present, a clearer understanding of how the splicing machinery functions awaits more detailed information about the nature and dynamics of the myriad of protein-protein and protein-RNA interactions formed within the spliceosome.
