ATP hydrolysis is not required for the chemistry of RNA splicing per se since the two transesterification reactions preserve the high-energy phosphate bonds. However, extensive ATP hydrolysis is required for the assembly and rearrangements of the spliceosome. Some of the additional proteins that make up the spliceosome use the energy of ATP hydrolysis to break existing RNA–RNA interactions to allow the formation of new ones. Each successful splice requires approximately 200 proteins, if we include those that form the snRNPs.
What is the purpose of these rearrangements? First, they allow the splicing signals on the pre-RNA to be examined by snRNPs several times during the course of splicing. For example, the U1 snRNP initially recognizes the 5ʹ splice site through conventional base-pairing; as splicing proceeds, these base pairs are broken (using the energy of ATP hydrolysis) and U1 is replaced by U6 (Figure 6–29). This type of RNA–RNA rearrangement (in which the formation of one RNA–RNA interaction requires the disruption of another) occurs several times during splicing and allows the spliceosomes to check and recheck the splicing signals, thereby increasing the overall accuracy of splicing. Second, the rearrangements that take place in the spliceosome create the active sites for the two transesterification reactions. These two active sites are created, one after the other, and only after the splicing signals on the pre-mRNA have been checked several times. This orderly progression ensures that splicing accidents occur only rarely.
One of the most surprising features of the spliceosome is the nature of the catalytic sites: they are formed by both protein and RNA molecules, although the RNA molecules catalyze the actual chemistry of splicing. In the last section of this chapter, we discuss in general terms the structural and chemical properties of RNA molecules that allow them to act as catalysts.
Once the splicing chemistry is completed, the snRNPs remain bound to the lariat. The disassembly of these snRNPs from the lariat (and from each other) requires another series of RNA–RNA rearrangements that require ATP hydrolysis, thereby returning the snRNAs to their original configuration so that they can be used again in a new reaction. At the completion of a splice, the spliceosome directs a set of proteins to bind to the mRNA near the position formerly occupied by the intron. Called the exon junction complex (EJC), these proteins mark the site of a successful splicing event and, as we shall see later in this chapter, influence the subsequent fate of the mRNA.
Other Properties of Pre-mRNA and Its Synthesis Help to Explain the Choice of Proper Splice Sites
As we have seen, intron sequences vary enormously in size, with some being in excess of 100,000 nucleotides. If splice-site selection were determined solely by the snRNPs acting on a preformed, protein-free RNA molecule, we would expect frequent splicing mistakes—such as exon skipping and the use of “cryptic” splice sites (Figure 6–30). The fidelity mechanisms built into the spliceosome to suppress errors, however, are supplemented by two additional strategies that further increase the accuracy of splicing. The first is a simple consequence of splicing being coupled to transcription. As transcription proceeds, the phosphorylated tail of RNA polymerase carries several components of the spliceosome (see Figure
Figure 6–29 One of the many rearrangements that take place in the spliceosome during pre-mRNA splicing.
This example comes from the yeast Saccharomyces cerevisiae, in which the nucleotide sequences involved are slightly different from those in human cells. The exchange of U1 snRNP for U6 snRNP occurs just before the first phosphoryltransfer reaction (see Figure 6–28). This exchange requires the 5ʹ splice site to be read by two different snRNPs, thereby increasing the accuracy of 5ʹ splice-site selection by the spliceosome.