134 7 Splicing and Alternative Splicing Impact on Gene Design
nuclear ribonucleoprotein particles (snRNPs). Each snRNP consists of an snRNA
(two in the case of U4/U6) and seven Sm proteins that form a ring-shaped struc-
ture (U6, as an exception, contains Sm-like proteins). Each snRNP contains
additionally a variable number of particle-specific proteins. Furthermore, a large
number of auxiliary proteins assemble co-transcriptionally on nascent pre-
mRNAs to accurately recognize the splice sites [5, 9–11].
The cis-acting pre-mRNA sequence elements help to define the splice sites and
mediate interactions between the pre-mRNA and components of the spliceo-
some [12–14]. The 5′ splice site interacts with the U1 snRNP via base pairing
between the splice site and the 5′ end of the U1 snRNA. The 3′ end is consecu-
tively recognized by several proteins, including non-snRNP factors like splicing
factor 1 (SF1), which binds to the branch point. The U2 auxiliary factor (U2AF),
a heterodimer consisting of a 65 and a 35 kDa subunit, binds the polypyrimidine
tract and the 3′ splice site. These factors form the early (E) complex. In a subse-
quent step, the E complex is joined by the U2 snRNP that binds to the branch
point forming the A complex. This structure is then bound by the preassembled
tri-snRNP consisting of the U5 and the U4/U6 snRNPs, generating the precata-
lytic B complex. The B complex undergoes major rearrangements in RNA–RNA
and RNA–protein interactions, leading to the destabilization of U1 and U4
snRNP binding. This catalytically activates the B complex to mediate the first
catalytic step of splicing and yields the C complex, which in turn catalyzes the
second step. The spliceosome then dissociates and is recycled for additional
rounds of splicing [5, 7, 11]. Lately several high-resolution structures of different
spliceosomal complexes from budding yeast and humans have been solved using
cryo-electron microscopy (e.g., [15–17]). These structures give unprecedented
insight on the architecture of the different complexes and aid our understanding
of the structural rearrangements that have to occur to complete one catalytic
cycle.7.2.4 Exon Definition
When the length of an intron exceeds 200–250 nucleotides, which is the case for
most introns in higher eukaryotes, early splicing complexes form across an exon
[18], a process called exon definition [19]. During exon definition, the U1 snRNP
binds to the 5′ splice site downstream of an exon and promotes the association of
U2AF with the polypyrimidine tract at the upstream 3′ splice site. This leads
subsequently to the recruitment of the U2 snRNP to the branch point upstream
of the exon. The complex is stabilized by the binding of additional proteins of the
serine/arginine (SR) protein family (see Section 7.5.2) to enhancer elements
within the exon [20, 21]. In addition, exon definition might be facilitated by pairs
of intronic enhancer elements flanking constitutive as well as alternatively spliced
exons [22].
Before proceeding to the splicing reaction, exon-defined complexes must be
converted to intron-defined complexes. This requires disruption of the cross-
exon interactions, followed by conversion into a cross-intron A complex, in
which a molecular bridge is formed from U2 to U1 bound to an upstream 5′
splice site [21, 23]. In an alternative assembly pathway, the tri-snRNP is already