Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA

Abstract

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25 degrees C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.

Figure 1

(A) Structural model of the td intron based on phylogeny and biochemical data (39,47). Base-paired elements are termed P1 to P10, joining regions between stems are termed J2/3–J8/7, and hairpin loops are numbered L1–L9.2. Thin black lines indicate long-range tertiary interactions. The splice sites (5′ and 3′ SS) are marked with an arrow. The deletion of the intron open reading frame (ORF) is also indicated in loop L6a. The intron sequence is displayed in upper case letters and exon sequences are shown as lower case letters. Arrows indicate the td mutants investigated in this work. (B) Schematic representation of the primer extension assay: reverse transcription products obtained from mRNA, cryptic RNA and pre-mRNA. Cryptic RNA results from an alternatively folded 5′ splice site (48,49).

Figure 2

Splicing of the base triple mutant tdC49U is sensitive to StpA. (A) Splicing of the base triple mutant tdC49U in the absence and presence of StpA and Cyt-18, respectively. Gel sample showing reverse transcription products corresponding to mRNA, cryptic RNA and pre-mRNA. (B) Quantification of four independent experiments. Values are given as (%) mRNA splicing and were calculated as described in Materials and Methods.

Figure 3

Splicing of mutants with a destabilized stem P3 is sensitive to StpA. (A) Schematic representation of investigated mutants. Notice that the mutation tdU912C causes a mismatch-destabilizing stem P3, whereas the double mutant tdA42G/U912C results in a stabilized P3 element which is also indicated by the T_m of the intron structure. (B) Splicing of mutants in stem P3 was assayed in the absence and presence of StpA and Cyt-18, respectively. Quantification of three independent experiments.

Figure 4

Splicing of a loop E mutant is increased in the presence of StpA. (A) Schematic representation of the loop E motif using the base pair annotation described in (50). The loop E mutant G905A is indicated. (B) First derivative of a UV absorbance profile for melting of RNA tertiary and secondary structures due to increasing temperature is shown for wild-type td intron (in blue), the tdG905A mutant in the loop E motif (in yellow) and the tdC865U mutant in P6 (in red). (C) Splicing of the loop E mutants tdG905A and tdC865U in the absence and presence of StpA and Cyt-18, respectively. Quantification of four independent experiments.

Figure 5

StpA can act as RNA chaperone and rescue destabilized td intron mutants at low temperature. (A) Splicing of wild-type td intron at either 37°C or 25°C in the absence or presence of StpA. Quantification of eight independent experiments. (B) Splicing activity of mutants U912C and C865U in the absence or presence of StpA at 25°C compared to the activity at 37°C. Quantification of two independent experiments.

Figure 6

In vitro splicing assays of wt and mutant pre-mRNA in the absence and presence of wild-type StpA and mutant G126V-StpA. Shown is the decrease of pre-mRNA with time. The indicated time points are 15, 30 and 45 s, and 1, 2, 5, 10, 30 and 60 min. The reactions were performed in the presence of 5 mM MgCl₂, 0.5 mM GTP, 50 mM Tris–HCl (pH 7.3) and 0.4 mM spermidine (see Materials and Methods). (A) Splicing of the td wt intron pre-RNA without added protein (full circles), with the addition of 1.4 μM wild-type StpA (full squares) or 1.4 μM G126V-StpA mutant (empty squares). (B) Splicing of the td C865U pre-RNA intron mutant without protein (full circles), with addition of 1.4 μM wild-type StpA (full squares) or 1.4 μM G126V-StpA mutant (empty squares). The graphs were calculated according to the equation given in Materials and Methods.