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

324 Chapter 6: How Cells Read the Genome: From DNA to Protein
splicing plays important roles in the development of cystic fibrosis, frontotemporal
dementia, Parkinson’s disease, retinitis pigmentosa, spinal muscular atrophy,
myotonic dystrophy, premature aging, and cancer. It has been estimated that
of the many point mutations that cause inherited human diseases, 10% produce
aberrant splicing of the gene containing the mutation.
The plasticity of RNA splicing also means that the cell can easily regulate the
pattern of RNA splicing. Earlier in this section we saw that alternative splicing can
give rise to different proteins from the same gene and that this is a common strategy
to enhance the coding potential of genomes. Some examples of alternative
splicing are constitutive; that is, the alternatively spliced mRNAs are produced
continuously by cells of an organism. However, in many cases, the cell regulates
the splicing patterns so that different forms of the protein are produced at different
times and in different tissues (see Figure 6–26). In Chapter 7, we return to this
issue to discuss some specific examples of regulated RNA splicing.
Spliceosome-Catalyzed RNA Splicing Probably Evolved from
Self-splicing Mechanisms
When the spliceosome was first discovered, it puzzled molecular biologists. Why
do RNA molecules instead of proteins perform important roles in splice-site
recognition and in the chemistry of splicing? Why is a lariat intermediate used
rather than the apparently simpler alternative of bringing the 5ʹ and 3ʹ splice sites
together in a single step, followed by their direct cleavage and rejoining? The
answers to these questions reflect the way in which the spliceosome has evolved.
As discussed briefly in Chapter 1 (and in more detail in the final section of this
chapter), it is likely that early cells used RNA molecules rather than proteins as
their major catalysts and that they stored their genetic information in RNA rather
than in DNA sequences. RNA-catalyzed splicing reactions presumably had critical
roles in these early cells. As evidence, some self-splicing RNA introns (that is,
intron sequences in RNA whose splicing out can occur in the absence of proteins
or any other RNA molecules) remain today—for example, in the nuclear rRNA
genes of the ciliate Tetrahymena, in a few bacteriophage T4 genes, and in some
mitochondrial and chloroplast genes. In these cases, the RNA molecule folds
into a specific three-dimensional structure that brings the intron/exon junctions
together and catalyzes the two transesterification reactions. A self-splicing intron
sequence can be identified in a test tube by incubating a pure RNA molecule that
contains the intron sequence and observing the splicing reaction. Because the
basic chemistry of some self-splicing reactions is so similar to pre-mRNA splicing,
it has been proposed that the much more involved process of pre-mRNA splicing
evolved from a simpler, ancestral form of RNA self-splicing.
RNA-Processing Enzymes Generate the 3ʹ End of Eukaryotic
mRNAs
We have seen that the 5ʹ end of the pre-mRNA produced by RNA polymerase II
is capped almost as soon as it emerges from the RNA polymerase. Then, as the
polymerase continues its movement along a gene, the spliceosome assembles
on the RNA and delineates the intron and exon boundaries. The long C-terminal
tail of the RNA polymerase coordinates these processes by transferring capping
and splicing components directly to the RNA as it emerges from the enzyme. In
this section, we shall see that, as RNA polymerase II reaches the end of a gene,
a similar mechanism ensures that the 3ʹ end of the pre-mRNA is appropriately
processed.
The position of the 3ʹ end of each mRNA molecule is specified by signals
encoded in the genome (Figure 6–34). These signals are transcribed into RNA as
the RNA polymerase II moves through them, and they are then recognized (as
RNA) by a series of RNA-binding proteins and RNA-processing enzymes (Figure
6–35). Two multisubunit proteins, called CstF (cleavage stimulation factor) and
CPSF (cleavage and polyadenylation specificity factor), are of special importance.
(A) NORMAL ADULT β-GLOBIN
RNA TRANSCRIPT
exon
1
exon
2
intron sequences
exon
3
normal mRNA is formed from three exons
(B) A SINGLE-NUCLEOTIDE CHANGE THAT
DESTROYS A NORMAL SPLICE SITE,
THEREBY CAUSING EXON SKIPPING
mRNA with exon 2 missing
(C) A SINGLE-NUCLEOTIDE CHANGE THAT
DESTROYS A NORMAL SPLICE SITE, THEREBY
ACTIVATING A CRYPTIC SPLICE SITE
mRNA with extended exon 3
(D) A SINGLE-NUCLEOTIDE CHANGE THAT
CREATES A NEW SPLICE SITE THEREBY CAUSING
A NEW EXON TO BE INCORPORATED
mRNA with extra exon inserted
between exon 2 and exon 3
Figure 6–33 Abnormal processing of
the β-globin primary RNA transcript in
humans with the disease β thalassemia.
In the examples shown, the disease (a
severe anemia due to aberrant hemoglobin
synthesis) is caused by splice-site
mutations found in the genomes of affected
patients. The dark blue boxes represent
the three normal exon sequences; the red
lines connect the 5ʹ and 3ʹ splice sites that
are used. In (B), (C), and (D), the light blue
MBoC6 m6.35/6.33
boxes depict new nucleotide sequences
included in the final mRNA molecule as a
result of the mutation denoted by the
black arrowhead. Note that when a
mutation leaves a normal splice site
without a partner, an exon is skipped (B)
or one or more abnormal cryptic splice
sites nearby is used as the partner site (C).
[Adapted in part from S.H. Orkin, in The
Molecular Basis of Blood Diseases
(G. Stamatoyannopoulos et al., eds.),
pp. 106–126. Philadelphia: Saunders,
1987.]