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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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Structural responses of the TL1/PK2 pseudoknot motif to antisense oligomers. (A) aiSHAPE principal. Hybridization of an interfering oligonucleotide (green) to one partner of the proposed RNA duplex increases acylation sensitivity of its base-paired counterpart. (B) Influence of LNA/DNA oligonucleotide (orange) hybridized to PK2 on chemical reactivity of TL1 residues. (C) The effect of LNA 1B hybridization to TL1 loop on acylation of PK2 nucleotides. The gray residues represent the formation of an extensive stop during reverse transcription at the place of LNA hybridization. The native traces (red) are compared with antisense-interfered DENV-MINI RNA (violet). Nucleotide positions exhibiting increased NMIA reactivity in the presence of given LNA are distinguished by orange brackets.
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gkt203-F3: Structural responses of the TL1/PK2 pseudoknot motif to antisense oligomers. (A) aiSHAPE principal. Hybridization of an interfering oligonucleotide (green) to one partner of the proposed RNA duplex increases acylation sensitivity of its base-paired counterpart. (B) Influence of LNA/DNA oligonucleotide (orange) hybridized to PK2 on chemical reactivity of TL1 residues. (C) The effect of LNA 1B hybridization to TL1 loop on acylation of PK2 nucleotides. The gray residues represent the formation of an extensive stop during reverse transcription at the place of LNA hybridization. The native traces (red) are compared with antisense-interfered DENV-MINI RNA (violet). Nucleotide positions exhibiting increased NMIA reactivity in the presence of given LNA are distinguished by orange brackets.

Mentions: To examine the TL1/PK2 tertiary interaction (13,16), antisense-interfered SHAPE (aiSHAPE) strategy has been applied (37). In this method, displacement of one strand of an RNA duplex by hybridizing an antisense oligonucleotide would disrupt long-distance interactions and would be characterized by enhanced NMIA sensitivity of the displaced nucleotides (Figure 3A).Figure 3.


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)

Structural responses of the TL1/PK2 pseudoknot motif to antisense oligomers. (A) aiSHAPE principal. Hybridization of an interfering oligonucleotide (green) to one partner of the proposed RNA duplex increases acylation sensitivity of its base-paired counterpart. (B) Influence of LNA/DNA oligonucleotide (orange) hybridized to PK2 on chemical reactivity of TL1 residues. (C) The effect of LNA 1B hybridization to TL1 loop on acylation of PK2 nucleotides. The gray residues represent the formation of an extensive stop during reverse transcription at the place of LNA hybridization. The native traces (red) are compared with antisense-interfered DENV-MINI RNA (violet). Nucleotide positions exhibiting increased NMIA reactivity in the presence of given LNA are distinguished by orange brackets.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt203-F3: Structural responses of the TL1/PK2 pseudoknot motif to antisense oligomers. (A) aiSHAPE principal. Hybridization of an interfering oligonucleotide (green) to one partner of the proposed RNA duplex increases acylation sensitivity of its base-paired counterpart. (B) Influence of LNA/DNA oligonucleotide (orange) hybridized to PK2 on chemical reactivity of TL1 residues. (C) The effect of LNA 1B hybridization to TL1 loop on acylation of PK2 nucleotides. The gray residues represent the formation of an extensive stop during reverse transcription at the place of LNA hybridization. The native traces (red) are compared with antisense-interfered DENV-MINI RNA (violet). Nucleotide positions exhibiting increased NMIA reactivity in the presence of given LNA are distinguished by orange brackets.
Mentions: To examine the TL1/PK2 tertiary interaction (13,16), antisense-interfered SHAPE (aiSHAPE) strategy has been applied (37). In this method, displacement of one strand of an RNA duplex by hybridizing an antisense oligonucleotide would disrupt long-distance interactions and would be characterized by enhanced NMIA sensitivity of the displaced nucleotides (Figure 3A).Figure 3.

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