Identification of antisense RNA stem-loops that inhibit RNA-protein interactions using a bacterial reporter system

Abstract

Many well-characterized examples of antisense RNAs from prokaryotic systems involve hybridization of the looped regions of stem-loop RNAs, presumably due to the high thermodynamic stability of the resulting loop-loop and loop-linear interactions. In this study, the identification of RNA stem-loops that inhibit U1A protein binding to the hpII RNA through RNA-RNA interactions was attempted using a bacterial reporter system based on phage lambda N-mediated antitermination. As a result, loop sequences possessing 7-8 base complementarity to the 5' region of the boxA element important for functional antitermination complex formation, but not the U1 hpII loop, were identified. In vitro and in vivo mutational analysis strongly suggested that the selected loop sequences were binding to the boxA region, and that the structure of the antisense stem-loop was important for optimal inhibitory activity. Next, in an attempt to demonstrate the ability to inhibit the interaction between the U1A protein and the hpII RNA, the rational design of an RNA stem-loop that inhibits U1A-binding to a modified hpII was carried out. Moderate inhibitory activity was observed, showing that it is possible to design and select antisense RNA stem-loops that disrupt various types of RNA-protein interactions.

Figure 1.

The strategy and design of a bacterial assay for detecting RNA loop–loop interactions that inhibit U1A–hpII-mediated antitermination complex formation. (A) A bacterial two-plasmid system based on phage λ N-mediated antitermination for detection of RNA–polypeptide interactions. (B) The secondary structure of the region of the RNA transcript containing the target U1 hpII and downstream RNA stem–loop library. The randomized nucleotides are indicated by N, and the regions corresponding to the PstI, BsrGI, and BamHI sites are shown in italic. (C) Possible mechanism for the disruption of antitermination complex formation by antisense RNA stem–loops.

Figure 2.

Native gel electrophoretic analysis of the intramolecular binding of the selected RNA stem–loop to boxA. (A) Schematic representation of the pseudoknot-like RNA folding by intramolecular RNA–RNA binding. (B) TBM gel (0.1 mM and 0.5 mM Mg²⁺) analysis of RNA substrates. All gels were run at 4°C;. Lane 1, 1-4; lane 2, 1-4 U₁₀₇; lane 3, 1-4 A₉; lane 4, 1-4 A₉/U₁₀₇; lane 5, 1-4 G₁₀₇C₁₀₈; lane 6, 1-4 G₈C₉; lane 7, 1-4 G₈C₉/G₁₀₇C₁₀₈; lane 8, 1-2; lane 9, 1-2 G₈C₉; lane 10, 1-8; lane 11, 1-8 G₈C₉.

Figure 3.

Gel mobility shift analysis of the intermolecular binding of the antisense stem–loop and boxA. (A) The secondary structure of the ‘inhibitor’ RNA stem–loop substrate. (B) The sequence of the boxA RNA substrates. (C) Gel shift analysis of the binding of ³²P-labeled wild-type and mutant SL1-4 RNAs with boxA and mutant RNAs on a TBM gel (0.1 mM Mg²⁺) at 4°C.

Figure 4.

The effect of the disruption of 1 and 2 bp in the upper stem region of the antisense RNA stem–loop of clone 1-4 and mutant constructs. (A) The secondary structure of the boxA and antisense stem–loop region of clone 1-4 and base substitutions investigated. The effect of the disruption of 1 and 2 bp in the upper stem of the antisense RNA on the antitermination activity of (B) clone 1-4, (C) clone 1-4 G₁₀₇C₁₀₈, (D) clone 1-4 G₈C₉ and (E) clone 1-4 G₈C₉/G₁₀₇C₁₀₈.

Figure 5.

Design of a modified U1 hpII RNA and cognate RNA stem–loop. The secondary structure of (A) HIV DIS, (B) U1 hpII, (C) U1 hpIIΔ^DIS and (D) the designed antisense stem–loop (aSL). (E) Antitermination activities of modified U1 hpII loop sequences.

Figure 6.

In vivo analysis of the inhibition of U1A protein binding to a modified hpII loop by the designed antisense stem-loop (aSL) and the effect of loop mutations. (A) The secondary structure of modified hpII-aSL region. (B) Antitermination activities of the designed constructs consisting of the the designed hpII–aSL, and aSL mutants.

Figure 7.

Gel mobility shift analysis of the inhibition of U1A protein binding to the modified hpII RNA (hpIIΔ^DIS) by the designed antisense RNA stem–loop (aSL). (A) U1A protein binding to hpII and hpIIΔ^DIS. (B) Binding of aSL and a mutant aSL (aSL 15C) to hpIIΔ^DIS. (C) Inhibition of U1A–hpIIΔ^DIS binding by aSL and aSL 15C.