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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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Mapping results for 3PSS and HPMut.The reagents are indicated by shape and color (see figure key). Only strong hits, with ≥2/3 the highest intensity band in a lane after subtraction of intensity from the control lane, are annotated. Pb2+ mapping results are taken from 4 min incubation time. For Pb2+, strong hits had ≥2/3 the intensity of A704 from PK, the strongest band observed under any condition at 4 min incubation time. Boxed residues are the center nucleotide of sites binding strongly (≥1/3 the highest intensity spot) and unambiguously to microarray probes (i.e. without alternative binding sites capable of forming stable duplexes within 3 kcal/mol of the optimal binding site). All folding buffers contained 10 mM Tris (pH7), 100 mM KCl. (A) Mapping results for 3PSS folded in 5 mM [Co(NH3)6]3+ (PK). (B) Mapping results for 3PSS folded without Mg2+ or [Co(NH3)6]3+ (HP). RNase A reactivity is not annotated because RNA is over-digested at the same enzyme concentration that yielded good results in PK and HPMut (Fig. S3). (C) Mapping results for HPMut folded in 10 mM Mg2+.
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pone-0038323-g003: Mapping results for 3PSS and HPMut.The reagents are indicated by shape and color (see figure key). Only strong hits, with ≥2/3 the highest intensity band in a lane after subtraction of intensity from the control lane, are annotated. Pb2+ mapping results are taken from 4 min incubation time. For Pb2+, strong hits had ≥2/3 the intensity of A704 from PK, the strongest band observed under any condition at 4 min incubation time. Boxed residues are the center nucleotide of sites binding strongly (≥1/3 the highest intensity spot) and unambiguously to microarray probes (i.e. without alternative binding sites capable of forming stable duplexes within 3 kcal/mol of the optimal binding site). All folding buffers contained 10 mM Tris (pH7), 100 mM KCl. (A) Mapping results for 3PSS folded in 5 mM [Co(NH3)6]3+ (PK). (B) Mapping results for 3PSS folded without Mg2+ or [Co(NH3)6]3+ (HP). RNase A reactivity is not annotated because RNA is over-digested at the same enzyme concentration that yielded good results in PK and HPMut (Fig. S3). (C) Mapping results for HPMut folded in 10 mM Mg2+.

Mentions: To study individual conformations, mapping was carried out on 3PSS with 100 mM KCl and no multivalent cations to favor HP, and with 100 mM KCl and 5 mM [Co(NH3)6]3+ to favor PK. HPMut was mapped in 100 mM KCl with 10 mM MgCl2. PK and HP/HPMut share structural motifs P1, P2, and junction J1/2 (Fig. 3). The differences between the two conformations are the P0 and P3′ motifs in PK and the P3 stem-loop in HP/HPMut. Enzymatic mapping used RNase T1 (cleaves after unpaired G), RNase A (cleaves after unpaired C and U), and RNase If (cleaves after any single stranded nucleotide). Chemical mapping used DMS (methylates N1 of A and N3 of C when unpaired), CMCT (modifies N3 of U and N1 of G when unpaired), and DEPC (modifies an exposed N7 of A) [33]. Pb2+ cleavage [34], [35] and SHAPE mapping [36] were used to identify flexible regions.


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)

Mapping results for 3PSS and HPMut.The reagents are indicated by shape and color (see figure key). Only strong hits, with ≥2/3 the highest intensity band in a lane after subtraction of intensity from the control lane, are annotated. Pb2+ mapping results are taken from 4 min incubation time. For Pb2+, strong hits had ≥2/3 the intensity of A704 from PK, the strongest band observed under any condition at 4 min incubation time. Boxed residues are the center nucleotide of sites binding strongly (≥1/3 the highest intensity spot) and unambiguously to microarray probes (i.e. without alternative binding sites capable of forming stable duplexes within 3 kcal/mol of the optimal binding site). All folding buffers contained 10 mM Tris (pH7), 100 mM KCl. (A) Mapping results for 3PSS folded in 5 mM [Co(NH3)6]3+ (PK). (B) Mapping results for 3PSS folded without Mg2+ or [Co(NH3)6]3+ (HP). RNase A reactivity is not annotated because RNA is over-digested at the same enzyme concentration that yielded good results in PK and HPMut (Fig. S3). (C) Mapping results for HPMut folded in 10 mM Mg2+.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369869&req=5

pone-0038323-g003: Mapping results for 3PSS and HPMut.The reagents are indicated by shape and color (see figure key). Only strong hits, with ≥2/3 the highest intensity band in a lane after subtraction of intensity from the control lane, are annotated. Pb2+ mapping results are taken from 4 min incubation time. For Pb2+, strong hits had ≥2/3 the intensity of A704 from PK, the strongest band observed under any condition at 4 min incubation time. Boxed residues are the center nucleotide of sites binding strongly (≥1/3 the highest intensity spot) and unambiguously to microarray probes (i.e. without alternative binding sites capable of forming stable duplexes within 3 kcal/mol of the optimal binding site). All folding buffers contained 10 mM Tris (pH7), 100 mM KCl. (A) Mapping results for 3PSS folded in 5 mM [Co(NH3)6]3+ (PK). (B) Mapping results for 3PSS folded without Mg2+ or [Co(NH3)6]3+ (HP). RNase A reactivity is not annotated because RNA is over-digested at the same enzyme concentration that yielded good results in PK and HPMut (Fig. S3). (C) Mapping results for HPMut folded in 10 mM Mg2+.
Mentions: To study individual conformations, mapping was carried out on 3PSS with 100 mM KCl and no multivalent cations to favor HP, and with 100 mM KCl and 5 mM [Co(NH3)6]3+ to favor PK. HPMut was mapped in 100 mM KCl with 10 mM MgCl2. PK and HP/HPMut share structural motifs P1, P2, and junction J1/2 (Fig. 3). The differences between the two conformations are the P0 and P3′ motifs in PK and the P3 stem-loop in HP/HPMut. Enzymatic mapping used RNase T1 (cleaves after unpaired G), RNase A (cleaves after unpaired C and U), and RNase If (cleaves after any single stranded nucleotide). Chemical mapping used DMS (methylates N1 of A and N3 of C when unpaired), CMCT (modifies N3 of U and N1 of G when unpaired), and DEPC (modifies an exposed N7 of A) [33]. Pb2+ cleavage [34], [35] and SHAPE mapping [36] were used to identify flexible regions.

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