148 7 Splicing and Alternative Splicing Impact on Gene Design
During the last years, a plethora of different riboswitch classes sensing a diverse
set of ligands has been discovered. In most cases the ligands are nucleobases,
amino acids, or coenzymes, but also ion- and second messenger-sensing ribos-
witches have been identified (reviewed in [225–227]).7.7.1 Regulation of Group I Intron Splicing in Bacteria
Recently two structurally different classes of riboswitches sensing the second
messenger cyclic diguanylate (c-di-GMP) were discovered [228, 229]. In Clo
stridium difficile, a class II c-di-GMP riboswitch was identified upstream of a
group I self-splicing intron [228]. Here, the start codon of the downstream
gene is engaged in base pairing interactions with sequences of the group I
intron structure. Furthermore, the intron contains an atypical 5′ splice site that
is partially sequestered by base pairing with an anti-5′ splice site sequence. In
the absence of c-di-GMP, guanosine triphosphate (GTP) cannot attack the
sequestered 5′ splice site, but attacks a site near the 3′ splice site, resulting in a
nonfunctional mRNA, with an accessible start codon, but lacking a ribosomal
binding site (rbs) (see Figure 7.3a, middle). Formation of the riboswitch struc-
ture in the presence of c-di-GMP leads to disruption of the anti-5′ splice site
stem. The correct 5′ splice site can now be attacked by GTP and the group I
intron removed completely. As a result, the start codon is not longer seques-
tered. In addition, a functional rbs is created by the joining of the exon
sequences (see Figure 7.3a, left side). Both events ultimately lead to gene
expression.
Apart from the allosteric activation of self-splicing, the c-di-GMP riboswitch
in C. difficile can regulate translation of the downstream gene in a second step
[230]. After removal of the group I intron, the upstream riboswitch lies next to
the newly created rbs. Access to the rbs is then regulated by binding of c-di-GMP
to the aptamer domain like in classical translation-controlling riboswitches (see
Figure 7.3, right side and bottom). To this date, this is the only example of a ribos-
witch regulating a self-splicing intron.7.7.2 Regulation of Alternative Splicing by Riboswitches in Eukaryotes
So far only one riboswitch class – the thiamine pyrophosphate (TPP) ribos-
witch – has been found in all three domains of life. In all identified cases, the
eukaryotic riboswitches are located in introns and regulate alternative splicing in
a TPP-dependent manner. Depending on the organism, the intronic sequences
containing the riboswitch are located in different parts of the pre-mRNA, and
alternative splicing subsequently triggers different downstream effects [231,
232]. In Aspergillus oryzae, a TPP riboswitch resides in an intron in the 5′ UTR
of a thiamin biosynthetic gene [233]. In Neurospora crassa, three TPP ribos-
witches have been identified [234]. Two of them reside in an intron in the 5′ UTR
(see Figure 7.3b). There, the intron encodes two 5′ splice sites. Under conditions
of low TPP concentration, the upstream 5′ splice site is used, leading to the com-
plete removal of the intronic sequence and high levels of gene expression. When
the TPP concentration is high, the downstream 5′ splice site mediates partial
retention of the intronic sequence. The retained sequence introduces a uORF,