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Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome.

Sztuba-Solinska J, Teramoto T, Rausch JW, Shapiro BA, Padmanabhan R, Le Grice SF - Nucleic Acids Res. (2013)

Bottom Line: Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2.Computer modeling implied that this motif might function as autonomous structural/regulatory element.In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

View Article: PubMed Central - PubMed

Affiliation: RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

ABSTRACT
The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5'-3' terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

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Secondary structure of the nucleotides 1–719 of DENV-MINI RNA. (A) Processed SHAPE reactivities of DENV-MINI RNA are presented as a function of nucleotide position. Red and orange notations are expected to fall in single-stranded regions, whereas bases indicated in black and green correspond predominantly to either double-stranded regions or putative tertiary interactions. (B) Three distinct DENV RNA domains have been annotated, namely, 5′–3′-UTRs, DBs and VR. Nucleotide positions are numbered every 20 nt. The abbreviations correspond to the following cis-acting RNA elements: UTR, untranslated region; SLA, stem–loop A; dsUAR, double-stranded upstream of AUG region; cHP, capsid hairpin; CS, cyclization sequences; DB, dumbbell; TL, top loop; SL, stem–loop. The pattern of Pb2+-induced hydrolysis obtained for interacting regions is indicated in the upper left insert.
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gkt203-F1: Secondary structure of the nucleotides 1–719 of DENV-MINI RNA. (A) Processed SHAPE reactivities of DENV-MINI RNA are presented as a function of nucleotide position. Red and orange notations are expected to fall in single-stranded regions, whereas bases indicated in black and green correspond predominantly to either double-stranded regions or putative tertiary interactions. (B) Three distinct DENV RNA domains have been annotated, namely, 5′–3′-UTRs, DBs and VR. Nucleotide positions are numbered every 20 nt. The abbreviations correspond to the following cis-acting RNA elements: UTR, untranslated region; SLA, stem–loop A; dsUAR, double-stranded upstream of AUG region; cHP, capsid hairpin; CS, cyclization sequences; DB, dumbbell; TL, top loop; SL, stem–loop. The pattern of Pb2+-induced hydrolysis obtained for interacting regions is indicated in the upper left insert.

Mentions: Reactivity of NMIA at each nucleotide position of DENV-MINI RNA is shown on Figure 1A and B and Supplementary Figure S1. The most reactive and thus least conformationally constrained residues have a reactivity >0.8 U and are depicted in red. Nucleotide positions with reactivity <0.2 U, indicative of fully base paired residues, are in black, whereas residues of intermediate reactivity are color-coded according to the key. Data shown are an average of at least three experiments.Figure 1.


Structural complexity of Dengue virus untranslated regions: cis-acting RNA motifs and pseudoknot interactions modulating functionality of the viral genome.

Sztuba-Solinska J, Teramoto T, Rausch JW, Shapiro BA, Padmanabhan R, Le Grice SF - Nucleic Acids Res. (2013)

Secondary structure of the nucleotides 1–719 of DENV-MINI RNA. (A) Processed SHAPE reactivities of DENV-MINI RNA are presented as a function of nucleotide position. Red and orange notations are expected to fall in single-stranded regions, whereas bases indicated in black and green correspond predominantly to either double-stranded regions or putative tertiary interactions. (B) Three distinct DENV RNA domains have been annotated, namely, 5′–3′-UTRs, DBs and VR. Nucleotide positions are numbered every 20 nt. The abbreviations correspond to the following cis-acting RNA elements: UTR, untranslated region; SLA, stem–loop A; dsUAR, double-stranded upstream of AUG region; cHP, capsid hairpin; CS, cyclization sequences; DB, dumbbell; TL, top loop; SL, stem–loop. The pattern of Pb2+-induced hydrolysis obtained for interacting regions is indicated in the upper left insert.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643606&req=5

gkt203-F1: Secondary structure of the nucleotides 1–719 of DENV-MINI RNA. (A) Processed SHAPE reactivities of DENV-MINI RNA are presented as a function of nucleotide position. Red and orange notations are expected to fall in single-stranded regions, whereas bases indicated in black and green correspond predominantly to either double-stranded regions or putative tertiary interactions. (B) Three distinct DENV RNA domains have been annotated, namely, 5′–3′-UTRs, DBs and VR. Nucleotide positions are numbered every 20 nt. The abbreviations correspond to the following cis-acting RNA elements: UTR, untranslated region; SLA, stem–loop A; dsUAR, double-stranded upstream of AUG region; cHP, capsid hairpin; CS, cyclization sequences; DB, dumbbell; TL, top loop; SL, stem–loop. The pattern of Pb2+-induced hydrolysis obtained for interacting regions is indicated in the upper left insert.
Mentions: Reactivity of NMIA at each nucleotide position of DENV-MINI RNA is shown on Figure 1A and B and Supplementary Figure S1. The most reactive and thus least conformationally constrained residues have a reactivity >0.8 U and are depicted in red. Nucleotide positions with reactivity <0.2 U, indicative of fully base paired residues, are in black, whereas residues of intermediate reactivity are color-coded according to the key. Data shown are an average of at least three experiments.Figure 1.

Bottom Line: Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2.Computer modeling implied that this motif might function as autonomous structural/regulatory element.In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

View Article: PubMed Central - PubMed

Affiliation: RT Biochemistry Section, HIV Drug Resistance Program, Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

ABSTRACT
The Dengue virus (DENV) genome contains multiple cis-acting elements required for translation and replication. Previous studies indicated that a 719-nt subgenomic minigenome (DENV-MINI) is an efficient template for translation and (-) strand RNA synthesis in vitro. We performed a detailed structural analysis of DENV-MINI RNA, combining chemical acylation techniques, Pb(2+) ion-induced hydrolysis and site-directed mutagenesis. Our results highlight protein-independent 5'-3' terminal interactions involving hybridization between recognized cis-acting motifs. Probing analyses identified tandem dumbbell structures (DBs) within the 3' terminus spaced by single-stranded regions, internal loops and hairpins with embedded GNRA-like motifs. Analysis of conserved motifs and top loops (TLs) of these dumbbells, and their proposed interactions with downstream pseudoknot (PK) regions, predicted an H-type pseudoknot involving TL1 of the 5' DB and the complementary region, PK2. As disrupting the TL1/PK2 interaction, via 'flipping' mutations of PK2, previously attenuated DENV replication, this pseudoknot may participate in regulation of RNA synthesis. Computer modeling implied that this motif might function as autonomous structural/regulatory element. In addition, our studies targeting elements of the 3' DB and its complementary region PK1 indicated that communication between 5'-3' terminal regions strongly depends on structure and sequence composition of the 5' cyclization region.

Show MeSH
Related in: MedlinePlus