Evidence of reciprocal tertiary interactions between conserved motifs involved in organizing RNA structure essential for internal initiation of translation

Abstract

Internal ribosome entry site (IRES) elements consist of highly structured RNA regions that determine internal initiation of translation. We have previously shown that the foot-and-mouth disease virus (FMDV) IRES contains a GNRA tetraloop spanning residues G178UAA181. Here we show that tertiary RNA interactions dependent on the GNRA motif determine the structural organization of the central domain. By using mutational analysis in combination with RNA probing, we have identified distant reciprocal interactions between the GNRA motif and the invariant region G240CACG244, termed motif A. Mutations in motif A caused a decrease in IRES activity as severe as the GUAG substitution in the GNRA motif. Substitutions in either GNRA or motif A sequences induced a common reorganization around the conserved R199AAA202 stem-loop, suggesting that the latter contributes to stabilize the GNRA-motif A interaction. This finding was also consistent with a significant increase in the efficiency of RNA-RNA interactions determined in gel shift assays using as probe the hairpin that contains the GNRA motif compared to a transcript encompassing the entire apical region of the central domain. Thus, we propose that the central domain of the FMDV IRES contains a structural conformation essential for IRES activity stabilized by a tertiary contact involving residues in the GNRA tetraloop and motif A conserved sequences.

FIGURE 1.

Secondary structure of FMDV IRES. Domains are numbered 1–5 or H–L; a dot is positioned every 10 residues. RNA structure of the central domain is depicted according to RNA probing analysis (Fernández-Miragall and Martínez-Salas 2003). Underlined symbols depict the substitutions in RAAA motif (A₁₉₉AAAG₂₀₃) to CGCCC, in GNRA motif (G₁₇₈UAA₁₈₁) to GUAG or UACG; motif A (G₂₄₀CACG₂₄₄) to ACGGC; and motif B (G₂₄₇ACU₂₅₀) to CGGG. Transcript stem 3 encompasses residues 86–299 with 151–227 deleted.

FIGURE 2.

RNase T2 probing of domain 3. RNA harboring the wild-type GUAA or UACG and GUAG substitution in the GNRA motif was incubated with RNase T2 in native (N) or denaturing (D) buffer conditions. A 5′-labeled primer was then used in a reverse transcriptase reaction, and cDNA products were subsequently analyzed on 6% acrylamide 7 M urea gels. A wild-type DNA sequence, prepared with the same oligonucleotide, was run in parallel to identify the RT stops. Residues are marked as the complementary sense sequence. Asterisks denote new attacks observed in the mutant RNAs relative to the GUAA sequence.

FIGURE 3.

(A) Relative activity of IRES mutants. Bicistronic constructs of the form CAT-IRES-luciferase carrying the indicated substitutions in motifs A or B were transfected in BHK-21 cells. GUAG and UCCG substitutions in the GNRA motif have been included for comparison. Relative IRES activity was calculated as the luciferase/CAT activity in each extract, made relative to that observed in the wild-type IRES. (B) Enzymatic probing of mutants in motifs A and B. RNA harboring the wild-type sequence or substitution in motifs A or B shown in A was incubated with RNase T2 in native (N) or denaturing (D) buffer and then analyzed by primer extension. Residues marked with asterisks denote new attacks relative to the wild-type sequence; open arrows denote lack of attack in the mutant RNAs.

FIGURE 4.

Reorganization of domain 3 RNA structure in substitution mutants of motifs A and B. (A) Secondary structure probing of the wild-type IRES using DMS and RNase A, T1, T2, and V1. (B) RNase T2 and DMS probing of motif A mutant. (C) RNase T2 and DMS probing of motif B mutant. Substituted residues are highlighted in red. For simplicity, only main changes relative to the wild type are depicted in the structure of mutants in motif A and B.

FIGURE 5.

RNA–RNA interactions dependent on the apical region of the central domain. (A) Schematic representation of domain 3 with indication of the regions encoded in transcripts used as probes used in gel shift assays. (B) 5′-end-labeled RAAA synthetic oligoribonucleotide (50 nM) was incubated in binding buffer with the indicated sense RNAs (400 nM), antisense (as), or tRNA; the last lane was incubated without Mg⁺². Transcript 3ABC corresponds to the apical region of domain 3, nt 151–227. Residues encompassing domains 1–2 and 4–5 are indicated in Figure1 ▶. RNA complexes were separated in native 6% acrylamide gels in TBM buffer. (C) Interaction of the FMDV GNRA hairpin is specific for the central domain. Gel shift analysis carried out with D3_160–196 transcript (50 nM) as probe and the different unlabeled FMDV IRES domains (400 nM). (D) Titration curve of transcript D3_160–196 interaction with the central domain. (E) Specificity of the interactions studied in gel shift assays. The antisense sequence of domain 1–2 (50 nM) was used as probe to interact with each of the indicated IRES sequences (400 nM) as indicated in panel B. The FMDV IRES was included as positive control. (F) Efficiency of RNA–RNA interaction within the central domain is increased by residues in the apical region. Gel shift performed with the enlarged version of the apical region D3_121–261 (50 nM) and the indicated transcripts (400 nM). The control antisense (c-as) RNA is described in Materials and Methods.

FIGURE 6.

Enzymatic probing of RAAA substitution mutant. (A) RNase T1 probing analyzed by primer extension. RNA harboring the CGCCC substitution in the RAAA motif was incubated with RNase T1. A filled arrow denotes changes with the pattern obtained in the wild-type domain 3 (Fernández-Miragall and Martínez-Salas 2003). (B) Direct RNase T1 cleavage pattern of [γ-³²P]ATP-5′-end-labeled CGCCC RNA, in comparison to the wild-type (AAAAG) RNA. Bands with the same electrophoretic mobility as cleavage fragments of the untreated transcript were not considered. Closed and empty arrows are used for new and lack of attack, respectively, relative to the wild-type sequence. (C) Reorganization of RNA structure in CGCCC mutant. Results from DMS attack have been represented on the secondary structure derived from RNase A, T1, and V1 digestion.

FIGURE 7.

Enzymatic and chemical probing of stem 3 RNA. (A) [γ-³²P]ATP-5′-end-labeled RNA was incubated with RNase T1 and analyzed on denaturing gel electrophoresis. (B) DMS and T2 treatment followed by primer extension analysis. (C) Schematic representation of stem 3 secondary structure. A thin line between residues G150 and U228 depicts the junction of the left and right arms in the truncated version of the central domain, stem 3. Symbols used are as in Figures 2 ▶, 4 ▶, and 6 ▶.