Trans-Activator Binding Site Context in RCNMV Modulates Subgenomic mRNA Transcription

Figures

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  Figure 1. RCNMV genome organization and subgenomic mRNA synthesis. (A) Structures of RCNMV RNA1 and RNA2 genomes. Nucleotide lengths are shown in kilobases (kb). Encoded proteins are shown as grey boxes with their corresponding sizes indicated in kilodaltons. The positions of the TA in RNA2 and the TABS in RNA1 are highlighted in yellow, and the TALS in RNA1 is highlighted in tan. The black dashed arrow signifies the interaction between complementary TA and TABS sequences. The location of the transcription initiation site of the sg mRNA in RNA1 is indicated by the angled arrow. (B) Subgenomic mRNA transcription from RNA1. The trans-TA–TABS interaction functions as an attenuating RNA signal for premature termination during minus (−)-strand synthesis of RNA1, resulting in a truncated (−)-strand, shown as a grey dashed line, which then serves as the template for transcription of the subgenomic mRNA.

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  Figure 2. RNA secondary structure models of the TA and TABS in dianthoviruses. (A) SHAPE-guided model of TA-containing region of RCNMV RNA2. The TA and AIL structures are denoted by grey and yellow shaded boxes, respectively. The green shading depicts the terminal loop of the TA that is complementary to the TABS. Relative reactivity of nucleotides are depicted as red, green, or black bases, indicating high, moderate, and low reactivity, respectively. Grey bases denote null reactivity values. (B) SHAPE-guided model of TABS-containing region of RCNMV RNA1. Relative nucleotide reactivities are as described in panel A. The TABS sequence is depicted by green shading and the TP-SL structure is shown in the dotted grey box. The position of the transcription initiation site for the subgenomic mRNA (sg) is shown as an angled arrow. (C) Comparative secondary structure analysis of RNA2 TA in RCNMV, SCNMV, and CRSV. The dotted arrow depicts two conformations of the CRSV TA structures with their corresponding computationally predicted free energies (kcal/mol). The red lines indicate mismatches in the TA–TABS interaction. (D) Comparative secondary structure analysis of the RNA1 TABS and TP-SL structures…

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  Figure 3. Mutational analysis of the TP-SL/TABS pairing in RCNMV RNA1. (A) Wild-type RCNMV RNA1 and mutants with nucleotide substitutions depicted in red with yellow highlighting. (B) RNA–RNA electrophoretic mobility shift assay of wt RNA1 and RNA1 mutants with wt RNA2 performed in 3% agarose gels stained with ethidium bromide. The position of RNA1–RNA2 complexes is indicted by an arrowhead. The contents of each lane are indicated above the gel and the percentages for shifted RNA1 are shown with SEM (n = 3). (C) Northern blot analysis of plus-strand genome accumulation from wt RNA1 and mutants transfected alone or with RNA2. RNA was isolated from protoplasts 22 h post transfection. The contents of each lane are indicated above the blot. The positions of the RNA1 and RNA2 genome segments, the subgenomic mRNA, and a stable degradation product of RNA1 (SR1f) are indicated on the left. Loading controls (rRNA) are also shown. In each coinfection, the level of sg mRNA accumulation was calculated relative to the accumulation level of its cognate RNA1. The relative levels of sg mRNA (Rel sg) and RNA2 (Rel RNA2) are shown below with SEM (n = 3).

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  Figure 4. Modifying the structural context of the TABS in RCNMV RNA1. (A) Structure of the TP-SL/TABS region in wt RCNMV RNA1. The potential partner sequence for the TABS is boxed in green, with existing base pairs indicated by the grey dotted line. The red dashed line defines the regions shown in (B). (B) RNA1 TP-SL/TABS mutants with nucleotide substitutions are depicted in red with yellow highlighting. Additional substitution mutations made in 182 (to generate 280 and 281) are circled in red. Intended base-pairing is shown by dotted lines for new (red) and existing (grey) partners. Amino acid changes are indicated to the left. (C) Northern blot analysis of wt plus-strand RNA1 and TP-SL/TABS mutants in plant protoplast transfections (n = 3). Loading controls (rRNA) are also shown. (D) Northern blot analysis of minus-strand RNA1 accumulation in plant protoplast transfections (n = 3). (E) Simplified depiction of the evolution of sg mRNA transcription from RNA1. Base pairing leading to sg mRNA transcription is depicted in red. See text for details.