Bacteria(a)
5 ′5 ′ 3 ′ 5 ′ 3 ′ 5 ′ 3 ′
ON OFF OFFGMPc-di-GMPc-di-++c-di-GMP+GTP +GTP+GTPORF 3 ′rbs ORFss ss3 ′(b) (c)Fungi+Plants5 ′ 3 ′ONOFFAAAAAATPP
ORFm^7 Gm^7 GORF
TPP
uORFuORFGU ORFGUGU AG+5 ′ONOFFAAAAAATPPm^7 Gm^7 GORF
TPP
ORFORFGUGU
AGss ORF rbs ssORFFigure 7.3 Splicing regulation by riboswitches. (a) In the bacterium C. difficile, a c-di-GMP binding riboswitch regulates splicing of
a group I intron. Depending on the presence of the ligand, different mRNAs are produced by alternative splicing. Left: Upon
c-di-GMP binding to the riboswitch, an otherwise sequestered 5′ splice site (indicated in red) becomes accessible to the cofactor
GTP, and the complete group I intron is removed. Therefore, joining of the exon sequences creates an accessible ribosomal binding
site (rbs) and the downstream gene can be expressed. Middle: In the absence of c-di-GMP, the correct 5′ splice site (indicated in
red) is inaccessible for GTP attack. GTP attack on a downstream site (indicated in pink) occurs, creating a truncated mRNA without
a ribosomal binding site. Therefore, gene expression is inhibited. Right: In very rare cases, the group I intron is correctly spliced in
the absence of c-di-GMP. Nevertheless, gene expression does not occur in the absence of c-di-GMP, as the newly created rbs is
sequestered within the riboswitch. In cases where the complete group I intron has been removed, subsequent c-di-GMP binding to
the aptamer domain of the riboswitch leads to structural rearrangements, rendering the rbs accessible. Gene expression can thus
be switched on or off, depending on the ligand binding state of the riboswitch. (b) In the filamentous fungus N. crassa, two genes
harbor a TPP riboswitch within an intron in their 5′ UTR. Both introns contain two 5′ splice sites. Top: In the absence of TPP, the
downstream 5′ splice site (pink) is sequestered by base pairing interactions with the free TPP aptamer domain. Consequently, the
upstream 5′ splice site (red) is used, leading to complete intron removal and subsequently to gene expression. Bottom: In the
presence of TPP, the aptamer domain binds its ligand, which renders the downstream 5′ splice site accessible to the spliceosome.
Thus, a part of the intron is retained after splicing, introducing a uORF, which inhibits gene expression. (c) In higher plants (e.g.,
Ara idopsis thaliana), TPP aptamer domains are found in introns within 3′ UTRs. There, gene expression is regulated by usage of
two different 3′ processing sites (diamonds). Top: In the absence of TPP, the 5′ splice site is sequestered by the aptamer domain,
leading to intron retention. 3′ processing occurs at the upstream site (red) encoded within the intron. This leads to a stable mRNA
with a short 3′ UTR and gene expression. Bottom: In the presence of TPP, the 5′ splice site is accessible and the upstream 3′
processing site is removed along with the intron. Usage of the downstream 3′ processing site (pink) leads to an mRNA with a long
3 ′ UTR. This mRNA is unstable, as long 3′ UTRs in plants trigger NMD. Therefore, gene expression is repressed. AAA = poly(A) tail,
c-di-GMP = cyclic diguanylate, GTP = guanosine triphosphate, m7G = 7-methylguanosine cap, ORF = open reading frame,
rbs = ribosomal binding site, ss = splice site, TPP = thiamine pyrophosphate, uORF = upstream open reading frame.