150 7 Splicing and Alternative Splicing Impact on Gene Design
which negatively affects gene expression. In the third case, the intron containing
the riboswitch is located in the main ORF of the gene. When the TPP level is low,
the intron is removed completely, resulting in gene expression [235]. TPP bind-
ing leads to incomplete intron removal by usage of downstream 5′ splice sites,
disrupting the main ORF by the introduction of frame shifts. So, in all three
cases, TPP leads to the downregulation of gene expression by modulation of 5′
splice site usage.
In higher plants, TPP riboswitches reside in the 3′ UTRs and control intron
retention by regulating the accessibility of the 5′ splice site (see Figure 7.3c)
[236, 237]. At low TPP concentrations, the 5′ splice site is inaccessible and a
stable mRNA with a short 3′ UTR is expressed. In the presence of high amounts
of TPP, the 5′ splice site is accessible and the intron in the 3′ UTR is removed.
Splicing of the intron also removes the major 3′ end processing site. As a result,
another downstream 3′ processing site is used, leading to an elongated 3′ UTR
that induces degradation by NMD.
In all cases studied, sequences within the aptamer domain of the TPP ribos-
witch base pair with splicing signals (usually the 5′ splice site), rendering them
inaccessible for the spliceosome. This sequestration of splicing signals then
triggers the usage of alternative 5′ splice sites, exon skipping, or intron reten-
tion. As the base pairing sequences in the TPP riboswitch are part of the ligand
binding pocket, binding of TPP leads to structural rearrangements, which ren-
ders the splicing signals accessible. Subsequent downstream events then
repress gene expression. This is achieved either by translational repression due
to uORFs in the 5′ UTR or by triggering NMD via PTCs or the length of the 3′
UTR.
The TPP riboswitch in N. crassa located in the intron of the main ORF is an
interesting exception (see preceding text). Here, the base pairing interactions in
the absence of TPP do not regulate alternative splicing by 5′ splice site sequestra-
tion, but facilitate intron removal [235]. This is achieved by a long-range interac-
tion between the aptamer domain and several conserved nucleotides downstream
of the 5′ splice site. It seems that, upon structure formation, reducing the effec-
tive distance between the 5′ and 3′ splice sites enhances splicing efficiency.
Until now, no eukaryotic homologs have been identified for the other ribos-
witch classes discovered in bacteria and archaea. Still, the report of a putative
arginine binding riboswitch, present in an intron in the 5′ UTR of an arginase in
the fungus Aspergillus nidulans, suggests that other eukaryotic riboswitches
might exist [238].7.8 Splicing and Synthetic Biology Contents ix
7.8.1 Impact of Introns on Gene Expression
Splicing is tightly linked with all stages of mRNA metabolism, including tran-
scription, mRNA processing, nuclear export, and translation. Intron sequences
may harbor transcriptional regulatory elements or affect DNA accessibility
by determining nucleosome arrangement, influence export processes, mRNA