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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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Location and structure of the 3′ splice site.(A) Segment 7 mRNA annotated with the splice sites (red arrow), UTRs (yellow box), and M1 and M2 open reading frames. M1 and M2 share the same start site, but M1 stops within the M2 ORF (at the black dotted line). Splicing combines the two green boxes to form the M2 open reading frame. (B) Predicted pseudoknot (PK) model with 3′ splice site annotation (red arrow), an SF2/ASF exonic splicing enhancer binding site in orange, and a polypyrimidine tract in blue. (C) Predicted hairpin (HP) model with same annotations as B. In order to isolate a single conformation, specific mutations were introduced to change nts 684 to 686 into A’s and to swap GC for CG at pairs 716–734 and 717–733 to forbid the pseudoknot and only allow the hairpin fold (HPMut).
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pone-0038323-g001: Location and structure of the 3′ splice site.(A) Segment 7 mRNA annotated with the splice sites (red arrow), UTRs (yellow box), and M1 and M2 open reading frames. M1 and M2 share the same start site, but M1 stops within the M2 ORF (at the black dotted line). Splicing combines the two green boxes to form the M2 open reading frame. (B) Predicted pseudoknot (PK) model with 3′ splice site annotation (red arrow), an SF2/ASF exonic splicing enhancer binding site in orange, and a polypyrimidine tract in blue. (C) Predicted hairpin (HP) model with same annotations as B. In order to isolate a single conformation, specific mutations were introduced to change nts 684 to 686 into A’s and to swap GC for CG at pairs 716–734 and 717–733 to forbid the pseudoknot and only allow the hairpin fold (HPMut).

Mentions: The influenza A virus possesses an eight segment (–) sense RNA genome, which codes for at least eleven proteins. Fragments of the influenza A coding RNA are predicted to have unusual thermodynamic stability, and also have suppressed third codon position variability. In combination with conserved base pairing, these results provided predictions of fragments likely to fold into functional structures [10]. One particularly interesting fragment (Fig. 1) includes the 3′ splice site of segment 7, as well as key residues of a binding site for the human SF2/ASF splicing factor [11] and a polypyrimidine tract that may bind other splicing factors such as U2AF65 [12], [13]. Segment 7 encodes the M1 matrix protein and three alternatively spliced products that share the 3′ splice site: the M2 protein, the small M3 polypeptide, and occasionally M4 [14]. Production of M2 is critical for uncoating of the viral genome and splicing of the M2 mRNA is temporally controlled [15].


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)

Location and structure of the 3′ splice site.(A) Segment 7 mRNA annotated with the splice sites (red arrow), UTRs (yellow box), and M1 and M2 open reading frames. M1 and M2 share the same start site, but M1 stops within the M2 ORF (at the black dotted line). Splicing combines the two green boxes to form the M2 open reading frame. (B) Predicted pseudoknot (PK) model with 3′ splice site annotation (red arrow), an SF2/ASF exonic splicing enhancer binding site in orange, and a polypyrimidine tract in blue. (C) Predicted hairpin (HP) model with same annotations as B. In order to isolate a single conformation, specific mutations were introduced to change nts 684 to 686 into A’s and to swap GC for CG at pairs 716–734 and 717–733 to forbid the pseudoknot and only allow the hairpin fold (HPMut).
© Copyright Policy
Related In: Results  -  Collection

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pone-0038323-g001: Location and structure of the 3′ splice site.(A) Segment 7 mRNA annotated with the splice sites (red arrow), UTRs (yellow box), and M1 and M2 open reading frames. M1 and M2 share the same start site, but M1 stops within the M2 ORF (at the black dotted line). Splicing combines the two green boxes to form the M2 open reading frame. (B) Predicted pseudoknot (PK) model with 3′ splice site annotation (red arrow), an SF2/ASF exonic splicing enhancer binding site in orange, and a polypyrimidine tract in blue. (C) Predicted hairpin (HP) model with same annotations as B. In order to isolate a single conformation, specific mutations were introduced to change nts 684 to 686 into A’s and to swap GC for CG at pairs 716–734 and 717–733 to forbid the pseudoknot and only allow the hairpin fold (HPMut).
Mentions: The influenza A virus possesses an eight segment (–) sense RNA genome, which codes for at least eleven proteins. Fragments of the influenza A coding RNA are predicted to have unusual thermodynamic stability, and also have suppressed third codon position variability. In combination with conserved base pairing, these results provided predictions of fragments likely to fold into functional structures [10]. One particularly interesting fragment (Fig. 1) includes the 3′ splice site of segment 7, as well as key residues of a binding site for the human SF2/ASF splicing factor [11] and a polypyrimidine tract that may bind other splicing factors such as U2AF65 [12], [13]. Segment 7 encodes the M1 matrix protein and three alternatively spliced products that share the 3′ splice site: the M2 protein, the small M3 polypeptide, and occasionally M4 [14]. Production of M2 is critical for uncoating of the viral genome and splicing of the M2 mRNA is temporally controlled [15].

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