Engineering of SD-adjacent quadruplexes

Translational modulation of gene expression via G-quadruplexes has been shown earlier by Hartig and co-workers. In that study, artificially designed sequences were placed around the ribosome binding site so that secondary structure formation inhibited interaction of the 16S rRNA with the eGFP-mRNA and hence initiation of translation. Repression of gene expression correlated with the thermodynamic stability of the G-quadruplex (161). A pronounced decrease of up to 96% of gene expression was observed in comparison to the wildtype, non-structured SD region in the artificial quadruplex system. Cis-repression and trans-activation of bacterial ribosome binding sites via secondary structure formation have been described in the context of engineered riboregulators (280). We were curious as to whether the opposite effect – activation of gene expression – could be accomplished by G-quadruplex formation in the 5’-UTR. In nature, e.g. the cold-sensing thermometer relies on RNA-regulatory mechanisms where a secondary structure formation liberates the SD region and hence activates gene expression (281). In order to implement a similar system based on quadruplex formation, another set of 5´-UTRs was designed. In these artificial designs the SD site can be masked by the formation of a long stem-loop structure in the mRNA (see Figure 3.7 A). This stem-loop structure contains a G-rich sequence strain potentially able to form a G-quadruplex. G-quadruplex formation competes with Watson-Crick base pairing in the stem (stem length ranging from 13 to 20 base pairs). An insertion of up to five single-nucleotide mismatches destabilizes the stem and should simplify quadruplex formation. The formed quadruplex ultimately dissolves the stem-loop structure so that the ribosome binding -site should become accessible for the ribosome, thus facilitating translation. These designs were investigated in a pBAD-eGFP reporter system. Predicted mfold structures (282) of the designs showing the mRNA region upstream of the start codon are depicted in Figure 3.8.

We chose the G3U quadruplex for our investigations as it is a short sequence that folds into a remarkably stable RNA G-quadruplex structure (283). In the first design the G3U quadruplex sequence was inserted 21 nt upstream of the eGFP start codon with full base pairing in the stem-loop structure. When comparing the quadruplex-bearing construct (G3U) to its control which should not be able to form a G-quadruplex (G3U ctrl) we observed a slight increase in gene expression (15%). Destabilization of the stem-loop structure by insertion of five mismatched base pairs allowed an easier quadruplex formation. For this construct (G3Umm) we observed a pronounced activation of gene expression of more than 100% compared to the control (G3Umm ctrl). Destabilization of the G-quadruplex by introduction of longer loops (G3CUmm) in the aforementioned construct still increased gene expression compared to the respective control (G3CUmm ctrl), but with less efficiency (89%, see Figure 3.7 B).

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However, addition of different G-quadruplex stabilizing compounds did not influence the gene expression levels (see Chapter 3.1.1.8). It is noteworthy that the mutations preventing G-quadruplex formation in the controls do not interfere with the complementary base pairing in the stem, but rather occur in the loop sequence (see Figure 3.8). Hence, base-pairing interactions within the hairpin structure do not differ between quadruplex constructs and controls. The similarity of eGFP-mRNA levels in G-quadruplex constructs and respective controls confirmed the regulation on the translational level (see Figure 3.7 C). Our results show that freeing the masked ribosome binding -site by the formation of a G-quadruplex in the mRNA could be a possible mechanism of translational regulation.

Figure 3.7:Artificial system comprising SD-adjacent quadruplexes.

A Mechanism suggested for enhancing gene expression via G-quadruplex formation. The red sequence is G-rich and able to form a quadruplex, but can also partly pair with the black sequence immediately 5’ of the SD region (blue). Access to the SD region can be blocked by formation of a stem loop structure. G-quadruplex formation leads to break up of the stem-loop structure and freeing of the SD site. Grey base pairs indicated mismatches introduced for facilitating quadruplex formation. B Modulation of eGFP expression. G3U:

GGGUGGGUGGGUGGG; G3U ctrl: GGGUGGGUGUGUGUG; G3CU mm: GGGCUGGGCUGGGCUGGG; G3CU mm ctrl: GGGCTGGGCTGTGCTGTG. C Analysis of eGFP mRNA levels by semi-quantitative RT-PCR for the engineered G-quadruplex constructs compared to their respective control. RNA levels were calculated relative to

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the expression of the genomically encoded ssrA gene. All experiments were performed in triplicates. Error bars represent standard deviations of three independent experiments, * indicates P<0.05, *** indicates P<0.0001. © Cell Press.

The demonstrated system of activating quadruplexes contradicts the results observed earlier for the G3T quadruplex inserted into the 5’-UTR on the sense strand 20 nt upstream of the start codon where a repression of gene expression was observed. This decrease was related to translational modulation, as mRNA levels did not change for G-quadruplex-bearing constructs compared to the respective controls in real-time PCR experiments. For the G3T inserted into the 5’-UTR on the sense strand 20 nt upstream of the start codon, the SD region should be easily accessible for the ribosome (see Figure 3.8 G for mfold prediction).

In the engineered system, the opposite effect was observed: G-quadruplexes inserted 21 nt upstream of the start codon activated gene expression. However, in this design the whole 5’-UTR was modified (nt composition and length between the SD site and the start codon) in order to mask the SD region if no quadruplex formation occurs. Hence, these two designs are not comparable and both results reflect possible influences of G-quadruplex sequences located close to the SD site. Furthermore, the overall gene expression of both the controls and the quadruplex constructs decreased in this system in comparison to the wt sequence, indicating an influence of sequence changes. In fact, changes in the 5’-UTR sequence have been found to alter the mRNA translation rate (284), as the 30S ribosomal complex appears to bind the upstream UTR (285,286). Therefore, the overall sequence and shape of the 5’-UTR has a strong impact on the actual effect of the G-quadruplex on gene expression.

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Figure 3.8: Predicted mRNA structures for the 5’ region of the artificial constructs.

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Predicted mRNA structure for the 5’ region of G-quadruplexes investigated for liberating SD site when blocked by a stem-loop structure. A G3U B G3Umm C G3CU D G3U ctrl E G3Umm ctrl F G3CU ctrl G Predicted structure of the G-quadruplex (G3T) inserted 20 nt in front of gene start in the pQE system. The SD site has the typical consensus sequence 5’-AGGAGGA-3’. Structure prediction according to mfold (http://mfold.rna.albany.edu/). © Cell Press.