7.5 Alternative Splicing in ammals 141snRNP to the 5′ splice site. Additionally, they recruit the U2AF heterodimer and
U2 snRNP to the 3′ splice site and help establish exon definition complexes (see
Section 7.2.4) [76, 134, 135]. Their activity is mediated by their RS domain and
the phosphorylation status (reviewed in [86]). The enhancing effect SR proteins
exert on exon inclusion is position dependent. Binding of SR proteins to intronic
regions can induce exon skipping [136]. Furthermore, binding of SR proteins to
exonic regions can have a differential impact on the inclusion of cassette exons.
Binding of SR proteins to ESEs within the cassette exon enhances its inclusion,
whereas binding to ESEs within the flanking constitutive exons promotes skip-
ping of the cassette exon [137, 138].
SR proteins can cooperate to promote exon inclusion. Different SR proteins
can recognize the same ESE and compensate for each other or act cooperatively
by binding to adjacent ESEs [69]. Additionally, SR proteins may form larger com-
plexes with other RS domain-containing proteins, such as the SR-related nuclear
matrix proteins SRm160 and SRm300, which are unable to bind RNA by them-
selves. These coactivators can form multiple interactions with snRNPs and
enhancer-bound SR proteins; thus they enhance activity through bridging inter-
actions between ESEs and spliceosomal components [139].
The splicing process can be inhibited by various mechanisms. Often, hnRNPs
like PTB or hnRNP A1 are involved. The simplest way of inhibition is sterically
blocking positive regulators. This happens when silencer elements are located
closely to splice sites or to splicing enhancer elements, so that splicing is inhib-
ited by blocking the access of snRNPs or positive regulatory factors. PTB, for
example, binds the polypyrimidine tract and therefore blocks binding of U2AF to
alternatively spliced exons [125]. Several other mechanisms by which PTB inhib-
its splicing have been elucidated [140]. It can inhibit U2AF binding also when
bound to exonic sequences [141]. PTB binding to ISSs can inhibit the transition
from an exon definition to an intron definition complex [142] or prevent interac-
tion of the U1 snRNP with other spliceosomal components [143]. Furthermore,
PTB might induce exon skipping by looping out exons flanked by intronic PTB
binding sites [144].
Like SR proteins, hnRNPs can cooperate to inhibit exon inclusion [68].
Recently, it was shown that inclusion of the cd45 exon 4 is repressed by hnRNP
L binding to an ESS. HnRNP L recruits hnRNP A1 and together the two hnRNPs
induce extended contacts of the U1 snRNP with exonic sequences, preventing
U6 snRNP contacts with the 5′ splice site and subsequent spliceosomal
catalysis [145].
Splicing of individual pre-mRNAs usually involves the integration of additive
and competitive signals from both splicing activating and repressing elements.
Along this line, SR proteins can induce exon inclusion by competing with repress-
ing hnRNPs. One example is the role of hnRNP A1 in the repression of exon 3 of
the HIV1 tat pre-mRNA. An ESS in exon 3 binds the repressor hnRNP A1 with
high affinity and inhibits splicing by propagating the binding of further hnRNP
A1 proteins toward the 3′ splice site. This propagative binding can be inhibited
by the binding of the SR protein SRSF1 to an upstream ESE, which then activates
splicing. Additionally, hnRNP A1 binds an ISS located upstream of exon 3,
thereby preventing binding of the U2 snRNP [7, 119, 146].