1. Introduction

22.14 Summary

Splicing accomplishes the removal of introns and the joining of exons into the mature sequence of RNA. There are at least four types of reaction, as distinguished by their requirements in vitro and the intermediates that they generate. The systems include eukaryotic nuclear introns, group I and group II introns, and tRNA introns. Each reaction involves a change of organization within an individual RNA molecule, and is therefore a cis-acting event.

Nuclear splicing follows preferred but not obligatory pathways. Only very short consensus sequences are necessary; the rest of the intron appears irrelevant. All 5′ splice sites are probably equivalent, as are all 3′ splice sites. We do not know how 5′ and 3′ sites are linked only in the proper pairs. The required sequences are given by the GT-AG rule, which describes the ends of the intron. The UACUAAC branch site of yeast, or a less well conserved consensus in mammalian introns, is also required. The reaction with the 5′ splice site involves formation of a lariat that joins the GU end of the intron via a 5′ V2′ linkage to the A at position 6 of the branch site. Then the 3′ VOH end of the exon attacks the 3′ splice site, so that the exons are ligated and the intron is released as a lariat. Both reactions are transesterifications in which bonds are conserved. Several stages of the reaction require hydrolysis of ATP, probably to drive conformational changes in the RNA and/or protein components. Lariat formation is responsible for choice of the 3′ splice site. Alternative splicing patterns are caused by protein factors that either stimulate use of a new site or that block use of the default site.

Nuclear splicing requires formation of a spliceosome, a large particle that assembles the consensus sequences into a reactive conformation. The spliceosome contains the U1, U2, U4/U6, and U5 snRNPs and some additional splicing factors. The U1, U2, and U5 snRNPs each contain a single snRNA and several proteins; the U4/U6 snRNP contains 2 snRNAs and several proteins. Some proteins are common to all snRNP particles. The snRNPs recognize consensus sequences. U1 snRNA base pairs with the 5′ splice site, U2 snRNA base pairs with the branch sequence, U5 snRNP acts at the 5′ splice site. When U4 releases U6, the U6 snRNA base pairs with U2, and this may create the catalytic center for splicing. An alternative set of snRNPs provides analogous functions for splicing the subclass of ATAC introns. The snRNA molecules may have catalytic-like roles in splicing and other processing reactions. In the nucleolus, two groups of snoRNAs are responsible for pairing with rRNAs at sites that are modified; group C/D snoRNAs indicate target sites for methylation, and group ACA snoRNAs identify sites where uridine is converted to pseudouridine.

Splicing is usually intramolecular, but some cases have been found of trans- (intermolecular) splicing. These reactions probably occur by spliceosome formation with the appropriate site sequences on each molecule.

Group II introns share with nuclear introns the use of a lariat as intermediate, but are able to perform the reaction as a self-catalyzed property of the RNA. These introns follow the GT-AG rule, but form a characteristic secondary structure that holds the reacting splice sites in the appropriate apposition.

Yeast tRNA splicing involves separate endonuclease and ligase reactions. The endonuclease recognizes the secondary (or tertiary) structure of the precursor and cleaves both ends of the intron. The two half-tRNAs released by loss of the intron can be ligated in the presence of ATP.

The termination capacity of RNA polymerase II has not been characterized, and 3′ ends of its transcripts are generated by cleavage. The sequence AAUAAA, located 11 V30 bases upstream of the cleavage site, provides the signal for both cleavage and polyadenylation. An endonuclease and the poly(A) polymerase are associated in a complex with other factors that confer specificity for the AAUAAA signal.