-1 Programmed ribosomal frameshifting in Class 2 umbravirus-like RNAs uses multiple long-distance interactions to shift between active and inactive structures and destabilize the frameshift stimulating element

Figures

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  Figure 1.. Class 2 ulaRNAs. A. Gene organization of Class 2 ulaRNAs. All ulaRNAs encode their own RdRp as a -1 PRF extension of the 5′ proximal ORF and all except CY1 have an additional ORF (ORF5). Monocot-infecting Class 2 ulaRNAs have an extra embedded ORF (ORF6) of different lengths. B. Maximum likelihood phylogenetic tree based on RdRp amino acid sequences. Branch numbers indicate bootstrap support in percentage out of 1000 replicates. The scale bar denotes amino-acid substitutions per site. Umbravirus PEMV2 was used as an outgroup and rooted to it. Dicot-infecting and monocot-infecting Class 2 ulaRNAs separate into different clades with the exception of parsley umbra-like virus (PULV), which occupies its own clade. CY1, CY1a and CY1b were isolated from greenhouse-grown citrus grafted with material from the same original infected Southern California limequat trees (34) and all lack ORF5. CY2 is closely related to CY1 and was isolated from hemp. C. Nucleotide sequence similarity among Class 2 ulaRNAs. The shown secondary structure of CY1 divides into three domains as indicated. The ‘Bridge’ that separates Domains 1 and 3 forms from the lower stem of Domain 2. The 3′ cap-independent…

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  Figure 2.. Conserved structure in the vicinity of the recoding region of Class 2 ulaRNAs. A. Structure shown is from CY1. Additional structures for some Class 2 ulaRNAs are shown in Supplementary Figure S2. Nucleotide colors denote SHAPE reactivity for full-length CY1 transcripts synthesized in vitro (33). Note that the SHAPE data does not support the lower stems (S2 and S1) of hairpin H2. Hairpin labels and the Bridge are described in legend to Figure 1C. Slippery site with three additional conserved nucleotides (circled) are in orange. Stop codon for the first ORF is in yellow with a red border. Numbers reflect nucleotide positions in CY1. B. Recoding region of umbravirus PEMV2. The structure on the left is from (32). The structure on the right is an alternative structure conserved in some umbraviruses. Nucleotides shaded in gray participate in a long-distance interaction with residues in the apical loop of the 3′ terminal hairpin.

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  Figure 3.. CY1 frameshifting requires a UGA termination codon. A. Alternative structure for CY1 H1/H2. The two structures shown are conserved in all Class 2 ulaRNAs (Supplementary Figure S3). SHAPE color coding of nucleotides and shading are as described in the caption to Figure 2. Note that SHAPE data more closely support the H1/H2 structure on the right. B. Full-length CY1 transcripts were altered to convert the UGA termination codon to either UAA or UAG, with and without additional alterations to maintain base-pairing of the termination codon in the alternative conformation of H1. Base changes are in red. C. In vitro translation of full-length WT CY1 (WT) or mutant transcripts in WGE. Data are averages from at least three independent experiments and are normalized to levels of WT translation. Standard deviations are shown. One-way ANOVA was performed using GraphPrism software. *, ** and *** are P values of 0.01, <0.01 and <0.0001, respectively.

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  Figure 4.. H2 and H3 in the CY1 FSE are critical for efficient frameshifting. A. CY1 structure in the vicinity of the recoding site with SHAPE structure probing data. Location of alterations in full-length CY1 transcripts are shown. Altered residues are in red. Other color shadings are as described in the caption to Figure 2A. Red arrows denote deletion end points. B. In vitro translation of full-length WT CY1 (WT) or mutant transcripts in WGE. Data are averages from at least three independent experiments and are normalized to levels of WT translation. Standard deviations are shown. One-way ANOVA was performed using GraphPrism software. *, ** and *** are P values of 0.01, <0.01 and <0.0001, respectively. PRF levels are normalized to WT CY1.

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  Figure 5.. Six proposed tertiary interactions with the CY1 FSE. CY1 structure is presented with SHAPE structure probing data. Other color shadings are as described in the caption to Figure 2A. PK1 (gray) pairs bases in the apical loop of H2 with either a sequence proximal to the 3′ end (PK1a) or local H2 internal sequences (PK1b). PK2 (green) pairs bases in the apical loop of H3 with bases in Domain 3 just past the Bridge (PK2a, dark green). C650 (circled) along with three downstream bases also pair with bases just upstream of the Bridge in Domain 1 (PK2b, light green). PK3 (blue) pairs bases in the apical loop of H4 with sequence on the 5′ side of H2 and has a second possible pairing partner that overlaps PK2b sequences (blue box). PK4 pairs bases in the hairpin H5 interior bulge with a sequence on the 3′ side of H2. All these tertiary interactions are conserved in all Class 2 ulaRNAs (Supplementary Figure S4).

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  Figure 6.. PK1a is important for efficient frameshifting. A. Proposed PK1a and PK1b tertiary interactions. For simplicity, H2 apical loop sequences, PK1b pairing sequences and PK1a pairing sequences are labeled [X], [Y] and [Z], respectively. Bases are colored by SHAPE structure probing. B. Table showing all Class 2 ulaRNA PK1 [X], [Y] and [Z] sequences. M or D denote whether the ulaRNA is monocot-infecting or dicot-infecting. Asterisk denotes that PULV occupies a different clade from the other dicot-infecting Class 2 members. The [Z] sequence is undetermined for SULV as the GenBank sequence terminates within the 3′ CITE. C. Mutations (in red) in [X] and [Z] sequences that were incorporated into full-length transcripts of CY1 and their effect on PK1a and PK1b. D. In vitro translation of full-length WT CY1 (WT) or mutant transcripts in WGE. Data are averages of at least three independent experiments and are normalized to levels of WT translation. Standard deviations are shown. One-way ANOVA was performed using GraphPrism software. *, ** and *** are P values of 0.01, <0.01 and <0.0001, respectively. E. WT and mutant CY1 minus-strand accumulation in vivo. A. thaliana protoplasts were…

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  Figure 7.. PK2a is more critical for frameshifting than PK2b. A. Proposed PK2a and PK2b tertiary interactions. Bases are colored by SHAPE structure probing. For simplicity, H3 apical loop sequences, PK2b pairing sequences and PK2a pairing sequences are labeled [X], [Y] and [Z]. B. Table showing all Class 2 ulaRNA PK2 [X], [Y] and [Z] sequences. With the possible exception of PULV, PK2a and PK2b interactions contain four base-pairs with one shared nucleotide (C650), and are found in identical locations in all Class 2 ulaRNAs. The nucleotide in red is C650 in the [X] sequence and pairing partners in the other two sequences. C. Mutations (in red and blue) that disrupt PK2a, PK2b or both. D. In vitro translation of full-length WT CY1 (WT) or mutant transcripts in WGE. Data are averages of at least three independent experiments and are normalized to levels of WT translation. Standard deviations are shown. One-way ANOVA was performed using GraphPrism software. *, ** and *** are P values of 0.01, <0.01 and <0.0001, respectively. PRF values are a percentage of the WT level.

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  Figure 8.. PK3 long-distance interaction is supported by compensatory mutations. A. Proposed PK3 tertiary interaction (in blue) between the apical loop of H4, located just upstream of a Y- shaped structure conserved in all ulaRNAs, and sequence on the 5′ side of H2 that includes the central symmetrical loop extending downward into S2. A second pairing partner for the H4 PK3 sequence is present in most Class 2 ulaRNAs overlaps the PK2b [Z] sequence just upstream of the Bridge (boxed in blue). Bases are colored by SHAPE structure probing. B. Table showing all Class 2 ulaRNA PK3 pairing partners in H2 and H4, as well as the possible additional pairing partner in the PK2b [Z] sequence (underlined sequence is involved in PK2b). No potential pairing partner for the H4 sequence is discernable for PULV in the PK2b [Z] location. Single asterisk denotes that PULV occupies a different clade from the other dicot-infecting Class 2 members. Double asterisks denotes that this sequence in H2 is in the S3 stem on the opposite site from the other PK3 interactions. C. Nucleotides are colored by SHAPE reactivity. Mutations (in red) that disrupt PK3 are shown. D. In vitro translation of full-length WT CY1 (WT)…

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  Figure 9.. PK4 long-distance interaction is supported by compensatory mutations. A. Proposed PK4 tertiary interaction (in red) between a bulge loop in H5, located just downstream of the Y-shaped structure, and sequence on the 3′ side of the H2 S2 stem. Unlike PK3, no additional pairing partner for the H5 sequence is discernable. B. Table showing all Class 2 ulaRNA PK4 pairing partners in H2 and H5. C. Nucleotides are colored by SHAPE reactivity. Mutations (in red) that disrupt PK4 are shown. D. In vitro translation of full-length WT CY1 (WT) or mutant transcripts in WGE. Data are averages of at least three independent experiments and are normalized to levels of WT translation. Standard deviations are shown. One-way ANOVA was performed using GraphPrism software. *, ** and *** are P values of 0.01, <0.01 and <0.0001, respectively. PRF values are a percentage of the WT CY1 level.

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  Figure 10.. Alternative structures for Class 2 ulaRNA FSEs. Conserved alternative structure is shown for CY1 and selected Class 2 ulaRNAs. These structures contain the alternative, extended form of H1 shown in Figure 3, right, and Supplementary Figure S3. Sequences that participate in tertiary interactions in the active structure are color coded: PK1b, gray; PK2a, dark green; PK2b, light green; PK3, blue; PK4, red. Nucleotides in CY1 are colored according to SHAPE reactivity, which offer better support for this structure than the structure shown in Figure 2. PK1b is compatible with this structure in all Class 2 ulaRNAs.

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  Figure 11.. Model for -1 PRF in Class 2 ulaRNAs. A. Frameshifting using the active FSE structure. B. Inactive structure promoting translation termination and conversion between inactive and active structures. See text for explanation.