Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

FROM DNA TO RNA
321
The Spliceosome Uses ATP Hydrolysis to Produce a Complex
Series of RNA–RNA Rearrangements
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
U1
exon 1 C A U U C A
ATP ADP
exon 1
5′ G U A U G U 3′
rearrangement
5′ GUAUG U 3′
GAGACA
U6
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.