Investigation of possible RNA G-quadruplex-interacting proteins

G-quadruplex forming sequences seem to play an important role in the regulation of gene expression. Therefore, it is assumed that the formation as

well as the unwinding of G-quadruplexes is tightly regulated within cells. While many proteins interacting with DNA G-quadruplexes have been identified, only a few proteins interacting with RNA G-quadruplexes have been found yet.

Among them are the two helicases DHX36 ¹⁹⁶ and more recent DHX9 ¹⁹⁷ which were shown to possess RNA G-quadruplex unwinding activity. Both helicases belong to the human DExH/D-box family of RNA helicases, indicating that members of this protein family might be involved in RNA G-quadruplex regulation. Therefore, further members of the human DExH/D-box family with RNA helicase activity, namely DHX29 ¹⁹⁸, DDX3X ¹⁹⁹, eIF4a1, eIF4a2 and eIF4a3 ²⁰⁰, were investigated for their ability to influence the MAPK2 RNA G-quadruplex formation. In addition, DHX36 was also tested for its potential to unwind the MAPK2 RNA G-quadruplex.

To study the ability of DHX36, DHX29, DDX3X, eIF4a1, eIF4a2 and eIF4a3 to unwind the MAPK2 RNA G-quadruplex within the 5´ UTR of an mRNA, HeLa 229 cells were firstly transfected with 10 nM siRNAs against the respective proteins 24 h before transfection with the reporter constructs. As reporter constructs the plasmid pcDNA5/FRT/TO-eGFP containing the MAPK2 G-quadruplex forming sequence at position 48 nt downstream of the 5´ UTR start or the corresponding control sequence and the plasmid pcDNA5/FRT/TO-mCherry were used. The level of reporter expression was measured 24 h after plasmid transfection of HeLa 229 cells via the Tecan infinite M200 fluorescence-reader (Fig. 3.1.4).

To proof the efficiency of the knock-down of possible RNA G-quadruplex-interacting proteins by siRNAs in HeLa 229 cells, the knock-down of DHX36 by siRNAs was exemplarily determined. Therefore, the expression of DHX36 after transfection of the cells with the respective siRNAs was examined via immunoblotting. For this purpose HeLa 229 cells were transfected with 10 nM siRNAs against DHX36 48 h before cell lysis. After separation of the cell lysate via SDS-gel electrophoresis, the siRNA-dependent knock-down of DHX36 was visualized by Western blot (Fig. 3.1.3).

3. Results and Discussion

Fig. 3.1.3: Knock-down of DHX36 by siRNAs in HeLa 229 cells. HeLa 229 cells were transfected with siRNAs (each final conc. of 10 nM) against DHX36 48 h before lysis. As control HeLa 229 cells transfected with nonsilencing siRNA (Ctrl) were used. Different amounts of total protein (10-80 µg) were loaded onto a 10% SDS-gel. Immunoblotting was performed using a polyclonal antibody against DHX36.

The Western blot analysis of HeLa 229 cells transfected with siRNAs against DHX36 revealed a siRNA-dependent reduction of the DHX36 protein expression (115 kDa) for all tested protein concentrations (Fig. 3.1.3). In the control sample (cells transfected with nonsilencing siRNA) DHX36 could be detected in correlation to the loaded protein concentrations. These results argue for an efficient knock-down of DHX36 by the siRNAs.

Fig. 3.1.4: Influence of the RNA helicases DHX36, DHX29, DDX3X, eIF4a1, eIF4a2 and eIF4a3 on the MAPK2 RNA G-quadruplex within the 5´ UTR of the reporter mRNA. HeLa 229 cells were transfected with 10 nM of the respective siRNAs 24 h before co-transfection with the reporter constructs pcDNA5/FRT/TO-eGFP containing the MAPK2 G-quadruplex forming

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sequence (GQ) or the corresponding control sequence (Ctrl) within the 5´ UTR of the reporter mRNA and pcDNA5/FRT/TO-mCherry. Transfection with the reporter construct was performed by using 100 ng plasmid/well in a 96 well format. Level of reporter expression was measured 24 h after plasmid transfection of HeLa 229 cells via fluorescence-reader. Numbers on the top of the gray bars show the relative inhibition of expression in comparison to the corresponding control (black bars) in percentage. The level of reporter expression of Ctrl was set to 1. The error bars represent the standard deviation of triplicates.

For the MAPK2 G-quadruplex forming sequence at position 48 nt downstream of the 5´ UTR (GQ), an inhibition of 25% of reporter expression was observed compared to the corresponding control (Ctrl) (Fig. 3.1.4). A further slight decrease in reporter expression could be only detected after the knock-down of DHX29, DDX3X or eIF4a2 by respective siRNAs. The knock-down of both DHX29 and eIF4a2 revealed a decrease of 29% in the reporter expression, which means a further decrease of only 4%. The knock-down of DDX3X resulted in a decrease of reporter expression of 33%. The knock-down of DHX36, whose ability for RNA G-quadruplex unwinding activity was already shown, did not show any effect. For the knock-down of eIF4a1 or eIF4a3 a slight increase in the reporter expression was detected.

Discussion:

In summary, the obtained results do not allow to make clear conclusions about the ability of the examined RNA helicases to influence the MAPK2 RNA G-quadruplex within the 5´ UTR. No distinct reduction of the reporter expression could be detected after knocking-down the RNA helicases DHX36, DHX29, DDX3X, eIF4a1, eIF4a2 and eIF4a3 by siRNAs. Although slight decreases in the reporter expression were observed after knock-down of DDX3X, eIF4a2 and DHX29, none of these results can be evaluated as differences in reporter expression with a view to the high standard deviations. It is also unclear why the knock-down of DHX36, which has already been shown to possess RNA G-quadruplex unwinding activity, failed to further reduce G-G-quadruplex-dependent reporter expression. Although the Western blot against DHX36 displayed an almost complete knock-down of DHX36 in HeLa 229 cells transfected with siRNAs, DHX36 might not be the major source of G-quadruplex unwinding activity in HeLa 229 cells and therefore its effect was not detectable in the used

3. Results and Discussion

system. It is also possible that unwinding of RNA G-quadruplexes is facilitated by several different enzymes and therefore knock-down of a single enzyme is not sufficient to detect a change in G-quadruplex-dependent reporter expression. Furthermore, it is also conceivable that, although DHX36 has RNA quadruplex unwinding activity, its unwinding activity is specific for certain G-quadruplexes. In Creascy et al. (2008) the G-quadruplex unwinding activity by DHX36 was detected by performing in vitro experiments with a sequence which folds into an intermolecular G-quadruplex ¹⁹⁶. Therefore, the herein tested quadruplex forming sequence, which should fold into an intramolecular G-quadruplex structure, might not be a relevant target of DHX36.

3.1.3 Investigation of possible RNA G-quadruplex-interacting small