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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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Chemical probing of PK2Flip mutant RNA. The description of the scheme follows the convention of the Figure 1 legend. The flipping mutation within PK2 is marked by a blue rectangle. The insert in the left top corner represents increased Pb2+-induced hydrolysis within PK2 and TL1 regions.
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gkt203-F4: Chemical probing of PK2Flip mutant RNA. The description of the scheme follows the convention of the Figure 1 legend. The flipping mutation within PK2 is marked by a blue rectangle. The insert in the left top corner represents increased Pb2+-induced hydrolysis within PK2 and TL1 regions.

Mentions: SHAPE analysis of PK2Flip DENV-MINI mutant RNA indicated major reactivity changes within the 5′ DB structure (Figure 4 and Supplementary Figures S4 and S5). The apical loop of the TL1-containing hairpin was rendered NMIA-sensitive with median reactivities for G470–U474 of 0.55, whereas the corresponding values for WT DENV-MINI were 0.05. The apex of the rightmost hairpin exhibited lower reactivity for residues G502–A506. In addition, the ‘flipping’ mutation of PK2 increased its susceptibility to acylation (C526–G530) validating disrupted TL1/PK2 base pairing. Other regions did not exhibit secondary structure rearrangements. Thus, data of Figure 4 and all subsequent illustrations representing RNA structures of DENV-MINI mutants present only the affected RNA motifs.Figure 4.


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)

Chemical probing of PK2Flip mutant RNA. The description of the scheme follows the convention of the Figure 1 legend. The flipping mutation within PK2 is marked by a blue rectangle. The insert in the left top corner represents increased Pb2+-induced hydrolysis within PK2 and TL1 regions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt203-F4: Chemical probing of PK2Flip mutant RNA. The description of the scheme follows the convention of the Figure 1 legend. The flipping mutation within PK2 is marked by a blue rectangle. The insert in the left top corner represents increased Pb2+-induced hydrolysis within PK2 and TL1 regions.
Mentions: SHAPE analysis of PK2Flip DENV-MINI mutant RNA indicated major reactivity changes within the 5′ DB structure (Figure 4 and Supplementary Figures S4 and S5). The apical loop of the TL1-containing hairpin was rendered NMIA-sensitive with median reactivities for G470–U474 of 0.55, whereas the corresponding values for WT DENV-MINI were 0.05. The apex of the rightmost hairpin exhibited lower reactivity for residues G502–A506. In addition, the ‘flipping’ mutation of PK2 increased its susceptibility to acylation (C526–G530) validating disrupted TL1/PK2 base pairing. Other regions did not exhibit secondary structure rearrangements. Thus, data of Figure 4 and all subsequent illustrations representing RNA structures of DENV-MINI mutants present only the affected RNA motifs.Figure 4.

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