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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

Native gels of 3PSS and HPMut under various conditions.(A) First lane is HPMut folded with 10 mM Tris (pH7), 100 mM KCl, and 10 mM MgCl2; the second lane is 3PSS with 10 mM Tris (pH7) and no monovalent or divalent ions. The remaining lanes are for 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing Mg2+ concentration (0, 2.5, 5.0, 7.5, 10, 15, and 25 mM). (B) First lane is 3PSS with 10 mM Tris (pH7), 100 mM KCl and no multivalent ions. The remaining lanes are 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing [Co(NH3)6]3+ concentration (0.002, 0.01, 0.02, 0.1, 2.5, and 5 mM).
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pone-0038323-g002: Native gels of 3PSS and HPMut under various conditions.(A) First lane is HPMut folded with 10 mM Tris (pH7), 100 mM KCl, and 10 mM MgCl2; the second lane is 3PSS with 10 mM Tris (pH7) and no monovalent or divalent ions. The remaining lanes are for 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing Mg2+ concentration (0, 2.5, 5.0, 7.5, 10, 15, and 25 mM). (B) First lane is 3PSS with 10 mM Tris (pH7), 100 mM KCl and no multivalent ions. The remaining lanes are 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing [Co(NH3)6]3+ concentration (0.002, 0.01, 0.02, 0.1, 2.5, and 5 mM).

Mentions: Native gels were run with a 63 nt fragment of an avian influenza A 3′ splice site (3PSS) from segment 7, alongside an artificial mutant construct (HPMut) that can fold into a hairpin but not a pseudoknot. Specifically, in HPMut, nucleotides 684-6 are changed to adenosines (Fig. 1B) and the two hairpin CG pairs at nucleotides 716–734 and 717–733 are swapped to make GC pairs (Fig. 1C); both changes forbid formation of the pseudoknot P0 helix while maintaining the hairpin. When the wild type sequence is folded in the presence of Mg2+, two bands are observed (Fig. 2A). The faster running of the two major bands observed for 3PSS (lanes 4–9 of Fig. 2A) migrates similar to HPMut (lane 1 of Fig. 2A). This suggests that the faster running band is the hairpin (HP) conformation of 3PSS and, by exclusion, that the slower running band is the pseudoknot (PK) conformation.


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)

Native gels of 3PSS and HPMut under various conditions.(A) First lane is HPMut folded with 10 mM Tris (pH7), 100 mM KCl, and 10 mM MgCl2; the second lane is 3PSS with 10 mM Tris (pH7) and no monovalent or divalent ions. The remaining lanes are for 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing Mg2+ concentration (0, 2.5, 5.0, 7.5, 10, 15, and 25 mM). (B) First lane is 3PSS with 10 mM Tris (pH7), 100 mM KCl and no multivalent ions. The remaining lanes are 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing [Co(NH3)6]3+ concentration (0.002, 0.01, 0.02, 0.1, 2.5, and 5 mM).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369869&req=5

pone-0038323-g002: Native gels of 3PSS and HPMut under various conditions.(A) First lane is HPMut folded with 10 mM Tris (pH7), 100 mM KCl, and 10 mM MgCl2; the second lane is 3PSS with 10 mM Tris (pH7) and no monovalent or divalent ions. The remaining lanes are for 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing Mg2+ concentration (0, 2.5, 5.0, 7.5, 10, 15, and 25 mM). (B) First lane is 3PSS with 10 mM Tris (pH7), 100 mM KCl and no multivalent ions. The remaining lanes are 3PSS with 10 mM Tris (pH7) and 100 mM KCl with increasing [Co(NH3)6]3+ concentration (0.002, 0.01, 0.02, 0.1, 2.5, and 5 mM).
Mentions: Native gels were run with a 63 nt fragment of an avian influenza A 3′ splice site (3PSS) from segment 7, alongside an artificial mutant construct (HPMut) that can fold into a hairpin but not a pseudoknot. Specifically, in HPMut, nucleotides 684-6 are changed to adenosines (Fig. 1B) and the two hairpin CG pairs at nucleotides 716–734 and 717–733 are swapped to make GC pairs (Fig. 1C); both changes forbid formation of the pseudoknot P0 helix while maintaining the hairpin. When the wild type sequence is folded in the presence of Mg2+, two bands are observed (Fig. 2A). The faster running of the two major bands observed for 3PSS (lanes 4–9 of Fig. 2A) migrates similar to HPMut (lane 1 of Fig. 2A). This suggests that the faster running band is the hairpin (HP) conformation of 3PSS and, by exclusion, that the slower running band is the pseudoknot (PK) conformation.

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