The Influenza A PB1-F2 and N40 Start Codons Are Contained within an RNA Pseudoknot

Influenza A is a negative-sense RNA virus with an eight-segment genome. Some segments encode more than one polypeptide product, but how the virus accesses alternate internal open reading frames (ORFs) is not completely understood. In segment 2, ribosomal scanning produces two internal ORFs, PB1-F2 and N40. Here, chemical mapping reveals a Mg²⁺-dependent pseudoknot structure that includes the PB1-F2 and N40 start codons. The results suggest that interactions of the ribosome with the pseudoknot may affect the level of translation for PB1-F2 and N40

Secondary Structure of a Conserved Domain in the Intron of Influenza A NS1 mRNA

<div><p>Influenza A virus is a segmented single-stranded (−)RNA virus that causes substantial annual morbidity and mortality. The transcriptome of influenza A is predicted to have extensive RNA secondary structure. The smallest genome segment, segment 8, encodes two proteins, NS1 and NEP, via alternative splicing. A conserved RNA domain in the intron of segment 8 may be important for regulating production of NS1. Two different multi-branch loop structures have been proposed for this region. A combination of <i>in vitro</i> chemical mapping and isoenergetic microarray techniques demonstrate that the consensus sequence for this region folds into a hairpin conformation. These results provide an alternative folding for this region and a foundation for designing experiments to probe its functional role in the influenza life cycle.</p></div

Stop-codon and C-terminal nonsense mutations are associated with a lower risk of cardiac events in patients with long QT syndrome type 1

In long QT syndrome type 1 (LQT1), the location and type of mutations have been shown to affect the clinical outcome. Although haploinsufficiency, including stop-codon and frameshift mutations, has been associated with a lower risk of cardiac events in patients with LQT1, nonsense mutations have been presumed functionally equivalent. The purpose of this study was to evaluate clinical differences between patients with nonsense mutations. The study sample comprised 1090 patients with genetically confirmed mutations. Patients were categorized into 5 groups, depending on mutation type and location: missense not located in the high-risk cytoplasmic loop (c-loop) (n = 698), which is used as reference; missense c-loop (n = 192); stop-codon (n = 67); frameshift (n = 39); and others (n = 94). The primary outcome was a composite end point of syncope, aborted cardiac arrest, and long QT syndrome-related death (cardiac events). Outcomes were evaluated using the multivariate Cox proportional hazards regression analysis. Standard patch clamp techniques were used. Compared to patients with missense non-c-loop mutations, the risk of cardiac events was reduced significantly in patients with stop-codon mutations (hazard ratio [HR] 0.57; 95% confidence interval [CI] 0.34-0.96; P = .035), but not in patients with frameshift mutations (HR 1.01; 95% CI 0.58-1.77; P = .97). Our data suggest that currents of the most common stop-codon mutant channel (Q530X) were larger than those of haploinsufficient channels (wild type: 42 ± 6 pA/pF, n = 20; Q530X+wild type: 79 ± 14 pA/pF, n = 20; P < .05) and voltage dependence of activation was altered. Stop-codon mutations are associated with a lower risk of cardiac events in patients with LQT1, while frameshift mutations are associated with the same risk as the majority of the missense mutations. Our data indicate functional differences between these previously considered equivalent mutation subtype

Diagram of the segment 8 (NS1/NEP) coding region.

<p>Numbering begins at the start of the NS1 open reading frame (ORF). White and shaded bars depict NS1 and NEP ORFs, respectively. Diagonal lines indicate the segment 8 intron and the blue box represents the 81–148 nt region.</p

Binding of nucleotides 81 to 148 of influenza A NS1 mRNA to isoenergetic probes on microarray.

a<p>- Center of binding site, where center is target RNA nucleotide complementary to third nucleotide from 5′-end of probe. Square brackets indicate possible alternative binding sites (if 5 or 6 nucleotides of probe bind) or region of binding (if predicted duplex has less than 5 canonical base pairs). Data in <i>italics</i> represent probes that bind in buffer II, but not in buffer I;</p>b<p>- LNA nucleotides are marked with superscript ^L, D represents 2,6-diaminopurine, nucleotides without superscript are 2′-O-methyl-nucleotides;</p>c<p>- S –strong binding, M – medium binding; <u>underline</u> – binding site with no ambiguity;</p>d<p>- buffer composition is 300 mM KCl, 10 mM MgCl₂, 10 mM Tris-HCl pH 7.0;</p>e<p>- buffer composition is 1 M KCl, 10 mM MgCl₂, 10 mM Tris-HCl pH 7.0;</p>f<p>- calculated by RNAstructure 4.6 program (for 1 M NaCl, assuming no structure of target RNA and unmodified probe), values correspond to binding sites listed in the first column;</p>g<p>- calculated for 100 mM NaCl buffer according to published equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Kierzek3" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Pasternak1" target="_blank">[38]</a>.</p><p>Probes listed underneath the double line have no perfect Watson-Crick match of the first five probe nucleotides to sequences in the target, but bind to target with at least one predicted GU wobble pair or in the case of site 114 have perfect complementarity to the target with only last five nucleotides of the probe. There are no complementary probes for sites: 129–131 and 137 on universal microarrays. Probes complementary to all other sites not listed in table do not bind strongly or moderately.</p

Structural models for nucleotides 81–148 of influenza A segment 8 (NS1/NEP).

<p>Numbering begins at the start of the NS1 open reading frame (ORF). Structural model A comes from Ilyinskii, et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Ilyinskii1" target="_blank">[12]</a> and the sequence is from strain A/PR/8/34 (H1N1), B is modeled <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Moss1" target="_blank">[9]</a> with RNAalifold <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Bernhart1" target="_blank">[18]</a>, and C is the MFE prediction from RNAstructure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Mathews1" target="_blank">[14]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Reuter1" target="_blank">[15]</a>. B and C have the consensus sequence for this region based on an alignment of 1017 unique sequences and can be found in many strains, including A/BrevigMission/1918 (H1N1), which caused the most deadly pandemic in history <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Johnson1" target="_blank">[16]</a>. RNAstructure free energy predictions at 37°C for sequences and structures A, B, and C are −7.1, −8.2, and −12.0 kcal/mol, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Reuter1" target="_blank">[15]</a>. These free energy predictions exclude the GC pair at 100/127 in structure B, because it is predicted not to form on the basis of thermodynamic parameters for secondary structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Mathews1" target="_blank">[14]</a>. Tertiary interactions may allow this pair to form or, alternatively, a C127/G131 base pair to form closing a UAA triloop. UAA triloops are common <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Lee1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Thulasi1" target="_blank">[43]</a> and can form tertiary interactions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Lescoute1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Ban1" target="_blank">[44]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Wimberly1" target="_blank">[45]</a>. Individual nucleotides are colored based on probabilities from RNAstructure partition function calculations as shown in the key <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Mathews2" target="_blank">[19]</a>. Colors of lines between nucleotides indicate type of conservation of pairing (see tables in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615.s001" target="_blank">Figure S1</a>). Strong modification sites for NMIA, CMCT, and DMS are indicated by colored-dots next to each reactive nucleotide. Red boxes indicate the center nucleotide of strongly binding iso-energetic microarray probes in 10 mM Tris-HCl, pH 7.0, 300 mM KCl, and 10 mM MgCl₂. Probe 127 could bind strongly to site 91, but it binds stronger than probe 91, indicating that site 127 is a true binding site for probe 127. Medium binding sites are not shown, but are centered at nucleotides 91 and 128. Light blue nucleotides next to structures B and C indicate positions in which sequence A differs from the consensus sequence. Solid red bars indicate the codons that were changed to GCG by Ilyinskii, et al. and led to down-regulation of NS1 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070615#pone.0070615-Ilyinskii1" target="_blank">[12]</a>. The chemical mapping data, particularly the strong reactivity of nucleotides 112 and 113, are consistent with structure C but not with structure B.</p