An intermolecular RNA triplex provides insight into structural determinants for the pseudoknot stimulator of -1 ribosomal frameshifting

Abstract

An efficient -1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote -1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several -1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of -1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the -1 PRF efficiency of this stimulator can be modulated by sequential removal of base-triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem-loop interactions and their relative positions to the helical junction play crucial roles for the -1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate -1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate -1 PRF. Furthermore, it suggests that -1 PRF, induced by an in-trans RNA via specific base-triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.

Figure 1.

Sequence, secondary structure and tertiary interaction comparisons among DU177 and several −1 PRF pseudoknots. (A) IBVm-PK (24). (B) MMTV-PK (12). (C) SRV-PK (25). (D) BWYV-PK (22). (E) Pea enation mosaic virus RNA1 pseudoknot (PEMV-1 PK) (26). (F) DU177 RNA pseudoknot (33). The nucleotides in DU177 are numbered according to those in ref. (33). The common AACAA sequences are highlighted by gray shadow, the unusual Hoogsteen AU base pairs are boxed, and the adenines stacking and tertiary interactions are represented by filled circles and dotted lines, respectively. (G) The 12% SDS–PAGE analysis of the −1 PRF assays of several viral RNA pseudoknots, including a minimum IBV-PK (IBV_m-PK), MMTV-PK, SRV-PK, BWYV-PK and DU177 RNA (22,25,29,32,33). Each pseudoknot was incorporated into a pRL-SV40-based reporter, assayed as described in the ‘Materials and Methods’, and the translated proteins corresponding to the 0-frame and −1 frame products were labeled as indicated. Please note that a negative control without insertion of a pseudoknot only showed background −1 PRF activity (data not shown).

Figure 2.

Both major- and minor-groove triple interactions in DU177 are required for efficient −1 PRF. (A) Illustration of mutagenesis scheme for the mutations in loops 1 and 2 of DU177. For each mutant, the nucleotide identities before and after mutation are typed in bold, linked by an arrow and labeled. In addition, mutants with multiple mutations are boxed. (B) The 12% SDS–PAGE analysis of −1 PRF assays of different loop mutation constructs. Individual mutant is indicated on top of the gel, with the translated proteins corresponding to the 0-frame and −1-frame products labeled as indicated. The calculated frameshift efficiencies (with standard deviation) are listed at the bottom of the gel and are the average of at least three repeated experiments. Please note that a p2luc reporter, with a stop codon inserted into the −1 frame of the N-terminal region of firefly luciferase, was used in these experiments and will thus generate a premature −1 frame protein product.

Figure 3.

CCC/GU RNA adopts the conformation of a pseudoknot. (A) Gel-mobility assay results for DU177 and CCC/GU RNA analyzed by 20% native gel. (B) Electrophoretic analysis of the enzymatic probing data of DU177. (C) Summary of the cleavage patterns for DU177. (D) Electrophoretic analysis of the enzymatic probing data of CCC/GU RNA. (E) Summary of the cleavage patterns for CCC/GU RNA. The enzymatic cleavage results were resolved in a 20% sequencing gel, with the first and eighth wells as alkaline hydrolysis ladder and control, respectively, whereas the last two wells represent guanine and pyrimidine assignment markers. The cleavage results after RNase T2 and V1 treatments, with increment of RNase concentration, are shown in wells 2–4 and 5–7, respectively. In addition, the assigned residues and the corresponding stem/loop regions are listed on the right and left sides of the gel, respectively. Please note that the residues different from each other are boxed for comparison between both RNAs. Finally, the extent of cleavage for each probe is defined as major or minor cut, and summarized in (B) and (D), with gray rhombuses representing RNase T2 cleavage and filled triangles representing RNase V1 cleavage. They are presented along the predicted secondary structures of both RNAs, with the five mutated loop nucleotides in CCC/GU RNA typed in gray.

Figure 4.

The −1 PRF assays for the stem–loop interaction dissection mutants of DU177. (A) Illustration of mutagenesis scheme for the mutations dissecting specific base–triple interactions. The mutants are designated as those in Figure 2A. (B) The 12% SDS–PAGE analysis of −1 PRF assays of different base–triple disruption mutants. Individual mutant is indicated on top of the gel, with the translated proteins corresponding to the 0-frame and −1-frame products labeled as indicated. The reporter construct is the same as Figure 2B, and the calculated −1 PRF efficiencies (with standard deviation) are listed at the bottom of the gel and are the average of at least three repeated experiments.

Figure 5.

DU177-mimicking intermolecular triplex can act as a stimulator of −1 PRF. (A) Schematic explanation for constructs used in the biomolecular pseudoknot approach. The base pairs formed intermolecularly are represented by filled circles, whereas tertiary base–triple interactions are shown by dotted lines. The nucleotides that will interfere with base triple formation in the bimolecular construct are typed in gray. Please note that the slippery site and spacer are not shown in the drawing, and a p2luc reporter, with a full-length firefly luciferase generated as the −1 frame product, was used in this experiment. (B) Results of −1 PRF assays of intermolecular major-groove base triple analysis. (C) Results of −1 PRF assays of intermolecular minor-groove base triple analysis. The designated asterisk indicates the estimated molecular ratios between the in-trans RNA (hTR3′ss, 171G3′ss and 172U3′ss) and the mRNA reporter (hTR5′hp and hTR5′hpL1c), and the translated proteins corresponding to the 0-frame and −1 frame products are also labeled.

Figure 6.

An intermolecular C-G·C major-groove base–triple interaction can replace an intermolecular U-A·U triple. The C-G·C major-groove base–triple interaction reconstituted in the bimolecular pseudoknot construct is typed in gray, and the other designations are the same as those in Figure 5.