132 7 Splicing and Alternative Splicing Impact on Gene Design
mRNA (splicing). Subsequently a complex machinery that deletes the interven-
ing sequences of the pre-mRNA was identified: the spliceosome. The discovery
that not all exons are included in the mature mRNA every time came as a further
surprise. This process was appropriately called alternative splicing and opened
up the possibility that one gene could code for more than one protein.
Alternative splicing is highly regulated during development and different
mRNA isoforms are important for determining the fate of different cell types and
tissues. Therefore, (alternative) splicing is viewed as an integral part of mRNA
maturation in eukaryotes, and aberrant splicing has not only been recognized to
be the causative agent of several hereditary diseases but also to drive cancer
progression.7.2 Nuclear Pre-mRNA Splicing in Mammals
7.2.1 Introns and Exons: A Definition
The average human gene contains eight exons with a mean length of 145 nucleo-
tides and introns more than ten times this size [3]. Cis-acting elements encoded
in the pre-mRNA provide the information that defines an intron (see Figure 7.1).
The 5′ splice site marks the beginning of the intron and includes the dinucleotide
GU encompassed within a larger, less conserved consensus sequence. The 3′ end
of the intron carries three conserved sequence elements. The branch point is
usually an adenosine located within a less conserved sequence element (branch
site), typically located 18–40 nucleotides upstream from the 3′ splice site. It is
followed by the polypyrimidine tract and a terminal AG dinucleotide at the
extreme 3′ end of the intron [4, 5]. The vast majority of introns contain the
canonical splice sites GU-AG (99%). However, other categories exist that occur
rarely, including the noncanonical splice sites GC-AG and AU-AC [6].7.2.2 The Catalytic Mechanism of Splicing
The splicing process consists of two consecutive transesterification reactions. In
the first step, the 5′ exon–intron junction is attacked by a free hydroxyl group
provided internally by the 2′ hydroxyl group from the branch point adenosine.
This leads to cleavage at the 5′ splice site and ligation of the 5′ end of the intron
to the 2′ hydroxyl group of the branch point adenosine. In the second step, the
free 3′ hydroxyl group of the released 5′ exon in turn attacks the phosphate at the
3 ′ intron–exon border. This results in ligation of the two exons and the release of
the intron in form of a lariat (reviewed in [5, 7, 8]).7.2.3 A Complex Machinery to Remove Nuclear Introns:
The Spliceosome
Splicing is catalyzed by the spliceosome, a large and highly dynamic macro-
molecular ribonucleoprotein complex that assembles on the intron-containing
pre-mRNA. The major spliceosome consists of the U1, U2, U4/U6, and U5 small