Aptazyme-dependent regulation of group I Intron-mediated splicing

3.4.1. Design and proposed mechanism

The artificial riboswitches constructed in this work so far were directly acting on the translational level, namely translation initiation [164, 180, 185], elongation [198] or ribosome stability. In this chapter, we designed a setup to control mRNA processing instead. Since in contrast to eukaryotes bacterial mRNA is usually not processed, we decided to implement a group I intron originating from Tetrahymena thermophila into the eGFP open reading frame (ORF). Upon self-splicing of the intron sequence, the eGFP exons would be reconnected and a functional gene product produced. If self-splicing is inhibited, however, a nonsense protein will be formed and reporter gene levels be reduced. We aimed for splicing control by inserting an HHAz into the group I intron scaffold thus destroying the intron structure upon ligand-induced self-cleavage, see figure 3.26.

Figure 3.26. Proposed mechanism for the aptazyme controlled self-splicing: The Tetrahymena thermophila group I intron is inserted into the ORF of the reporter gene eGFP. Only upon self-splicing would the functional eGFP mRNA sequence be recovered and eGFP eventually expressed. Activating the aptazyme by the external trigger TPP would result in self-cleavage prior to self-splicing and thus in inhibition of reporter gene expression.

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Figure 3.27. Secondary structure map of the Tetrahymena thermophila intron (Tth.L1925). Insertion sites of the HHR (depicted in grey) are shown as red boxes, splice sites are denoted with grey arrowheads. Intron sequence is written in black letters, eGFP in green.

So far, only aptamers alone have been used to control splicing. Insertion of the aptamer in proximity to sites within the intron which are essential for splicing resulted in cloaking of these sites upon addition of the ligand and finally reduced splicing efficacy. This design was shown to work for the 3’-splice site in vitro [89], and for the branching point sequence [93] and 5’-splice site [90] in Escherichia coli and yeast, respectively. Moreover, Ellington and co-workers developed a theophylline-dependent group I intron by attaching the theophylline aptamer to P6.

67 Addition of theophylline led to conformational changes stabilizing the catalytically active structure [108]. The splicing mechanism and structure of Tetrahymena thermophila group I intron is well characterized: Bound by the intron, an exogenous G performs a nucleophilic attack on the 5’-splice site. Subsequently, a conformational change induces a second cleavage reaction at the 3’-splice site resulting in exon ligation and intron release [206, 207]. The group I intron itself consists of ten helical regions [207] where P4 – P6 are forming rapidly the inner core followed by a slow assembly of P7 – P9 [208], see figure 3.27.

In order to construct an aptazyme-control intron, we first inserted the Tetrahymena thermophila group I intron into the reporter gene eGFP. Only efficient splicing will allow eGFP expression while a nonsense protein will be achieved if splicing is inhibited. Next, we had to test the influence of introducing the HHR scaffold on splicing activity. Therefore, we incorporated an inactivated HHR at different positions into the intron. If the HHR was not interfering with the catalytically active intron structure ligation efficacy should not be affected. Activation of the HHR, however, should prevent correct intron folding and eventually decrease eGFP expression, see figure 3.26. The HHR variants were incorporated into stem P2, P6, P8 and P10 of the intron, see figure 3.27. Attachment of the inactive HHR to P2 resulted in the complete inactivation of splicing (see figure 3.28 B) which is in accordance with structural data showing the loop region of P2 being involved in tertiary interactions with P8 [207]. Inserting the inactivated HHR at the other three positions still allowed for efficient reporter gene expression. This implies that the catalytic active intron structure is not disturbed by the HHR scaffold at the respective positions. Activation of these HHR variants by a G to A point mutation in the catalytic core proofed position P10 to be suited best for constructing an artificial riboswitch since it showed an inactivation ratio of 3.5, while P6 and P8 showed only ratios of 1.7 and 2.1 respectively, see figure 3.28 B. An explanation for the low inactivation ratio if attached to P6 could be the fast folding kinetics. As mentioned above, P4 to P6 are folded rapidly [208], probably exceeding the cleavage kinetics of the HHR.

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Figure 3.28. Insertion of the HHR at different positions in the group I intron. A) Sequence and secondary structure of the inserted HHR. Inactivating A to G point mutation in the catalytic core [156] is shown as boxed nucleotides. B) eGFP expression after insertion of the inactivated (white bars) and active (black bars) HHR at the corresponding position. Numbers on top of the bars represent the ratio of eGFP expression of the inactivated / active HHR variant.

3.4.2. Conclusion

In summary, we were able to control self-splicing of the Tetrahymena thermophila group I intron in E. coli by an HHR. In a next step, stem III can be again replaced by an aptamer in order to obtain a ligand-controllable group I Intron. Initial experiments with the TPP aptamer and the connection sequence from the mRNA context were promising but could not be repeated. Therefore, another in vivo screening for an optimized connection sequence between the HHR and the TPP domain will be performed.

To our best knowledge, they would represent the only way to control splicing by the addition of the non-toxic vitamin B1 (thiamine) in vivo. More importantly, group I introns have been shown to work efficiently in trans, thus enabling the linking of RNAs in vivo [209-211] upon addition of an external ligand.

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