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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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Related in: MedlinePlus

Proposed 3D model of truncated DENV-MINI RNA. The internally deleted DENV-MINI RNA is depicted (A) in its entirety or (B) highlighting the proximal 5′- and 3′-UTRs. Specific RNA domains are color-coded as shown in the key. The cis-acting motifs are labeled as follows: SLA TL, stem–loop A top loop; SLA SSL, stem–loop A side stem–loop; 3′ SL, 3′ stem–loop; dsUAR, double-stranded upstream of AUG region; PK, pseudoknot; TL, top loop. For clarity, the remainder of the RNA molecule is colored in gray.
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gkt203-F7: Proposed 3D model of truncated DENV-MINI RNA. The internally deleted DENV-MINI RNA is depicted (A) in its entirety or (B) highlighting the proximal 5′- and 3′-UTRs. Specific RNA domains are color-coded as shown in the key. The cis-acting motifs are labeled as follows: SLA TL, stem–loop A top loop; SLA SSL, stem–loop A side stem–loop; 3′ SL, 3′ stem–loop; dsUAR, double-stranded upstream of AUG region; PK, pseudoknot; TL, top loop. For clarity, the remainder of the RNA molecule is colored in gray.

Mentions: A 3D structural model of the DENV-MINI RNA was generated using RNAComposer, a fully automated server that predicts 3D structural models from RNA secondary structure (40) (Figure 7). As the server does not accept sequences longer than 500 nt, a 474-nt derivative sequence was created by deleting the VR domain and closing the remaining short helical region (A190–U194 and A443–U448) with a -GAGA- tetraloop. A dot-bracket notation generated by the RNAstructure software was adjusted to account for this internal deletion, as well as the predicted TL1/PK2 pseudoknot, and subsequently provided to RNAComposer. Ten 3D RNA models were generated and analyzed, taking into account their secondary structure topology, sequence homology, structure resolution and free energy. In addition, the quality of predicted models was evaluated using the MolProbity tool (24). The best of these models was manually adjusted to correct for an unlikely fold within PK1, and the affected regions were subjected to energy minimization (Accelrys Discovery Studio v3.5) to create the structure of Figure 7.Figure 7.


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)

Proposed 3D model of truncated DENV-MINI RNA. The internally deleted DENV-MINI RNA is depicted (A) in its entirety or (B) highlighting the proximal 5′- and 3′-UTRs. Specific RNA domains are color-coded as shown in the key. The cis-acting motifs are labeled as follows: SLA TL, stem–loop A top loop; SLA SSL, stem–loop A side stem–loop; 3′ SL, 3′ stem–loop; dsUAR, double-stranded upstream of AUG region; PK, pseudoknot; TL, top loop. For clarity, the remainder of the RNA molecule is colored in gray.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt203-F7: Proposed 3D model of truncated DENV-MINI RNA. The internally deleted DENV-MINI RNA is depicted (A) in its entirety or (B) highlighting the proximal 5′- and 3′-UTRs. Specific RNA domains are color-coded as shown in the key. The cis-acting motifs are labeled as follows: SLA TL, stem–loop A top loop; SLA SSL, stem–loop A side stem–loop; 3′ SL, 3′ stem–loop; dsUAR, double-stranded upstream of AUG region; PK, pseudoknot; TL, top loop. For clarity, the remainder of the RNA molecule is colored in gray.
Mentions: A 3D structural model of the DENV-MINI RNA was generated using RNAComposer, a fully automated server that predicts 3D structural models from RNA secondary structure (40) (Figure 7). As the server does not accept sequences longer than 500 nt, a 474-nt derivative sequence was created by deleting the VR domain and closing the remaining short helical region (A190–U194 and A443–U448) with a -GAGA- tetraloop. A dot-bracket notation generated by the RNAstructure software was adjusted to account for this internal deletion, as well as the predicted TL1/PK2 pseudoknot, and subsequently provided to RNAComposer. Ten 3D RNA models were generated and analyzed, taking into account their secondary structure topology, sequence homology, structure resolution and free energy. In addition, the quality of predicted models was evaluated using the MolProbity tool (24). The best of these models was manually adjusted to correct for an unlikely fold within PK1, and the affected regions were subjected to energy minimization (Accelrys Discovery Studio v3.5) to create the structure of Figure 7.Figure 7.

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