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The 3' splice site of influenza A segment 7 mRNA can exist in two conformations: a pseudoknot and a hairpin.

Moss WN, Dela-Moss LI, Kierzek E, Kierzek R, Priore SF, Turner DH - PLoS ONE (2012)

Bottom Line: In the two conformations, the splice site and other functional elements exist in very different structural environments.In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop.The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Center for RNA Biology, University of Rochester, Rochester, New York, United States of America.

ABSTRACT
The 3' splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3' splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.

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Related in: MedlinePlus

Pb2+ mapping results.(A) Gel images of PK (5 mM [Co(NH3)6]3+), HP (no multivalent ions), and HPMut (10 mM Mg2+) incubated with 1 mM Pb(OAc)2, 10 mM Tris (pH 7), and 100 mM KCl for 0, 0.5, 1, 2, 4, 8, 15, 30, and 60 min. (B) integrated band densities at each nucleotide normalized to the strongest band observed under any condition (A704 from PK) at 4 min incubation time. RNA structure is annotated below each graph where paired nucleotides that form complementary helical strands are indicated by colored boxes.
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pone-0038323-g004: Pb2+ mapping results.(A) Gel images of PK (5 mM [Co(NH3)6]3+), HP (no multivalent ions), and HPMut (10 mM Mg2+) incubated with 1 mM Pb(OAc)2, 10 mM Tris (pH 7), and 100 mM KCl for 0, 0.5, 1, 2, 4, 8, 15, 30, and 60 min. (B) integrated band densities at each nucleotide normalized to the strongest band observed under any condition (A704 from PK) at 4 min incubation time. RNA structure is annotated below each graph where paired nucleotides that form complementary helical strands are indicated by colored boxes.

Mentions: In 5 mM [Co(NH3)6]3+, where PK dominates, the P0 stem region, which incorporates the 3′ splice site, is not reactive (Fig. 3A). Of the three nucleotides that bridge P0 and P1, A687 is modified by SHAPE and A688 is modified by DMS. There are no strong hits on the P1 hairpin. Reactivity is clustered in the J1/2 junction and the 5′ end of the P3′ hairpin. The J1/2 junction is cleaved by RNase If, Pb2+ and modified by SHAPE and DMS. The P3′ hairpin is quite sensitive to enzymatic cleavage, but less so towards small molecules. RNases If, T1, and A cleave along the 5′ end of P3′, whereas the loop region is modified by DEPC at A724 and A730. PK is largely protected from Pb2+ cleavage (Fig. 4A,B). After incubation in Pb(OAc)2 for 4 min, strong cleavage only occurs at the J1/2 junction, particularly at A704 (Fig. 4B). Medium cleavage occurs at the loop of P3′ (Fig. 4 and Fig. S1A). Even after 60 min of incubation, PK is un-reactive outside these regions (Fig. 4A,B).


The 3' splice site of influenza A segment 7 mRNA can exist in two conformations: a pseudoknot and a hairpin.

Moss WN, Dela-Moss LI, Kierzek E, Kierzek R, Priore SF, Turner DH - PLoS ONE (2012)

Pb2+ mapping results.(A) Gel images of PK (5 mM [Co(NH3)6]3+), HP (no multivalent ions), and HPMut (10 mM Mg2+) incubated with 1 mM Pb(OAc)2, 10 mM Tris (pH 7), and 100 mM KCl for 0, 0.5, 1, 2, 4, 8, 15, 30, and 60 min. (B) integrated band densities at each nucleotide normalized to the strongest band observed under any condition (A704 from PK) at 4 min incubation time. RNA structure is annotated below each graph where paired nucleotides that form complementary helical strands are indicated by colored boxes.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369869&req=5

pone-0038323-g004: Pb2+ mapping results.(A) Gel images of PK (5 mM [Co(NH3)6]3+), HP (no multivalent ions), and HPMut (10 mM Mg2+) incubated with 1 mM Pb(OAc)2, 10 mM Tris (pH 7), and 100 mM KCl for 0, 0.5, 1, 2, 4, 8, 15, 30, and 60 min. (B) integrated band densities at each nucleotide normalized to the strongest band observed under any condition (A704 from PK) at 4 min incubation time. RNA structure is annotated below each graph where paired nucleotides that form complementary helical strands are indicated by colored boxes.
Mentions: In 5 mM [Co(NH3)6]3+, where PK dominates, the P0 stem region, which incorporates the 3′ splice site, is not reactive (Fig. 3A). Of the three nucleotides that bridge P0 and P1, A687 is modified by SHAPE and A688 is modified by DMS. There are no strong hits on the P1 hairpin. Reactivity is clustered in the J1/2 junction and the 5′ end of the P3′ hairpin. The J1/2 junction is cleaved by RNase If, Pb2+ and modified by SHAPE and DMS. The P3′ hairpin is quite sensitive to enzymatic cleavage, but less so towards small molecules. RNases If, T1, and A cleave along the 5′ end of P3′, whereas the loop region is modified by DEPC at A724 and A730. PK is largely protected from Pb2+ cleavage (Fig. 4A,B). After incubation in Pb(OAc)2 for 4 min, strong cleavage only occurs at the J1/2 junction, particularly at A704 (Fig. 4B). Medium cleavage occurs at the loop of P3′ (Fig. 4 and Fig. S1A). Even after 60 min of incubation, PK is un-reactive outside these regions (Fig. 4A,B).

Bottom Line: In the two conformations, the splice site and other functional elements exist in very different structural environments.In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop.The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Center for RNA Biology, University of Rochester, Rochester, New York, United States of America.

ABSTRACT
The 3' splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3' splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site.

Show MeSH
Related in: MedlinePlus