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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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TL1/PK2 pseudoknot interaction in DENV-MINI RNA. (A) SHAPE-predicted hairpin-type pseudoknot interaction comprises two helical regions, including base paring between PK2 (G526–C530) and TL1 residues (G470–U474), as well as the stem of the leftmost hairpin (C462–G466 and C478–G482), and three single-stranded loops: A468–G470, U474–C476 and C516–C525. (B) The 2D map of TL1/PK2 interaction forming H-type pseudoknot generated by PseudoViewer3 (32).
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gkt203-F2: TL1/PK2 pseudoknot interaction in DENV-MINI RNA. (A) SHAPE-predicted hairpin-type pseudoknot interaction comprises two helical regions, including base paring between PK2 (G526–C530) and TL1 residues (G470–U474), as well as the stem of the leftmost hairpin (C462–G466 and C478–G482), and three single-stranded loops: A468–G470, U474–C476 and C516–C525. (B) The 2D map of TL1/PK2 interaction forming H-type pseudoknot generated by PseudoViewer3 (32).

Mentions: With regard to the 3′-UTR, SHAPE provided information on the 3′ SL structure, obscured only at the extreme 3′ terminus because of the presence of hybridized primer (Figure 1B and Supplementary Figure S1). The 3′ SL displayed NMIA sensitivity at the apical loop (A674–C680) and insensitivity at its leftmost side. The fact that this large 79-nt stem–loop was followed with reactive stretch of 3 nt (C655–U657) and unreactive dsUAR region (nucleotides A81–G96 and nucleotides C638–U654), enforced the proposed conformation. Previous phylogenetic comparative studies suggested that the 3′-UTR enclosed two almost identical DBs that might assume a pseudoknot conformation with their downstream complementary regions (13,16). SHAPE data confirmed the presence of both, and in addition provided detailed insight into their secondary structure. The 3′ DB contained a 6-bp unreactive stem (U536–G541 and C598–A603) and two hairpins on opposing ends (G547–U571 and G584–C597). The apical loop of the leftmost hairpin incorporated the highly reactive TL2 sequence (G558–U562). The reactivity of TL2, combined with the fact that its downstream complementary partner PK1 (A613–C617) was involved in formation of 5′–3′ CS region, would likely prevent the previously suggested TL2/PK1 pseudoknot interaction (13,16). Regarding the 5′ DB, it likewise contained an unreactive stem (G449–G454 and C511–C516) and two hairpins on opposing ends (C462–G482 and G497–C510). Here, however, the apical loop of the leftmost hairpin incorporating the TL1 sequence (G467–G477), although predicted to be single-stranded, harbored mainly unreactive nucleotides. The fact that its complementary partner PK2 was likewise unreactive and enclosed within single-stranded loop (G526–C530) was strongly suggestive of a TL1/PK2 base pairing interaction (Figure 2) (13,16). In addition, both DBs comprised reactive bulges (A491–C496 and A578–A583) and moderately NMIA-sensitive purine-rich internal loops. One of the internal loops of the 3′ DB (G572–A575) was insensitive to modification as it contained a -GGAA- motif. Such -GNRA- tetraloops represent a unique fold, which include a trans sugar edge/Hoogsteen edge G∙A pair between the first and the last residues, a hydrogen bond between the 2′-OH of G and the N7 of A and 3′ stacking of all tetraloop bases except the G. This architecture would account for lack of reactivity towards NMIA (31).Figure 2.


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)

TL1/PK2 pseudoknot interaction in DENV-MINI RNA. (A) SHAPE-predicted hairpin-type pseudoknot interaction comprises two helical regions, including base paring between PK2 (G526–C530) and TL1 residues (G470–U474), as well as the stem of the leftmost hairpin (C462–G466 and C478–G482), and three single-stranded loops: A468–G470, U474–C476 and C516–C525. (B) The 2D map of TL1/PK2 interaction forming H-type pseudoknot generated by PseudoViewer3 (32).
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Related In: Results  -  Collection

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

gkt203-F2: TL1/PK2 pseudoknot interaction in DENV-MINI RNA. (A) SHAPE-predicted hairpin-type pseudoknot interaction comprises two helical regions, including base paring between PK2 (G526–C530) and TL1 residues (G470–U474), as well as the stem of the leftmost hairpin (C462–G466 and C478–G482), and three single-stranded loops: A468–G470, U474–C476 and C516–C525. (B) The 2D map of TL1/PK2 interaction forming H-type pseudoknot generated by PseudoViewer3 (32).
Mentions: With regard to the 3′-UTR, SHAPE provided information on the 3′ SL structure, obscured only at the extreme 3′ terminus because of the presence of hybridized primer (Figure 1B and Supplementary Figure S1). The 3′ SL displayed NMIA sensitivity at the apical loop (A674–C680) and insensitivity at its leftmost side. The fact that this large 79-nt stem–loop was followed with reactive stretch of 3 nt (C655–U657) and unreactive dsUAR region (nucleotides A81–G96 and nucleotides C638–U654), enforced the proposed conformation. Previous phylogenetic comparative studies suggested that the 3′-UTR enclosed two almost identical DBs that might assume a pseudoknot conformation with their downstream complementary regions (13,16). SHAPE data confirmed the presence of both, and in addition provided detailed insight into their secondary structure. The 3′ DB contained a 6-bp unreactive stem (U536–G541 and C598–A603) and two hairpins on opposing ends (G547–U571 and G584–C597). The apical loop of the leftmost hairpin incorporated the highly reactive TL2 sequence (G558–U562). The reactivity of TL2, combined with the fact that its downstream complementary partner PK1 (A613–C617) was involved in formation of 5′–3′ CS region, would likely prevent the previously suggested TL2/PK1 pseudoknot interaction (13,16). Regarding the 5′ DB, it likewise contained an unreactive stem (G449–G454 and C511–C516) and two hairpins on opposing ends (C462–G482 and G497–C510). Here, however, the apical loop of the leftmost hairpin incorporating the TL1 sequence (G467–G477), although predicted to be single-stranded, harbored mainly unreactive nucleotides. The fact that its complementary partner PK2 was likewise unreactive and enclosed within single-stranded loop (G526–C530) was strongly suggestive of a TL1/PK2 base pairing interaction (Figure 2) (13,16). In addition, both DBs comprised reactive bulges (A491–C496 and A578–A583) and moderately NMIA-sensitive purine-rich internal loops. One of the internal loops of the 3′ DB (G572–A575) was insensitive to modification as it contained a -GGAA- motif. Such -GNRA- tetraloops represent a unique fold, which include a trans sugar edge/Hoogsteen edge G∙A pair between the first and the last residues, a hydrogen bond between the 2′-OH of G and the N7 of A and 3′ stacking of all tetraloop bases except the G. This architecture would account for lack of reactivity towards NMIA (31).Figure 2.

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