Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries

While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein

On improvement in ejection fraction with iron chelation in thalassemia major and the risk of future heart failure

<p>Abstract</p> <p>Background</p> <p>Trials of iron chelator regimens have increased the treatment options for cardiac siderosis in beta-thalassemia major (TM) patients. Treatment effects with improved left ventricular (LV) ejection fraction (EF) have been observed in patients without overt heart failure, but it is unclear whether these changes are clinically meaningful.</p> <p>Methods</p> <p>This retrospective study of a UK database of TM patients modelled the change in EF between serial scans measured by cardiovascular magnetic resonance (CMR) to the relative risk (RR) of future development of heart failure over 1 year. Patients were divided into 2 strata by baseline LVEF of 56-62% (below normal for TM) and 63-70% (lower half of the normal range for TM).</p> <p>Results</p> <p>A total of 315 patients with 754 CMR scans were analyzed. A 1% absolute increase in EF from baseline was associated with a statistically significant reduction in the risk of future development of heart failure for both the lower EF stratum (EF 56-62%, RR 0.818, p < 0.001) and the higher EF stratum (EF 63-70%, RR 0.893 p = 0.001).</p> <p>Conclusion</p> <p>These data show that during treatment with iron chelators for cardiac siderosis, small increases in LVEF in TM patients are associated with a significantly reduced risk of the development of heart failure. Thus the iron chelator induced improvements in LVEF of 2.6% to 3.1% that have been observed in randomized controlled trials, are associated with risk reductions of 25.5% to 46.4% for the development of heart failure over 12 months, which is clinically meaningful. In cardiac iron overload, heart mitochondrial dysfunction and its relief by iron chelation may underlie the changes in LV function.</p

Gifsy-1 Prophage IsrK with Dual Function as Small and Messenger RNA Modulates Vital Bacterial Machineries

While an increasing number of conserved small regulatory RNAs (sRNAs) are known to function in general bacterial physiology, the roles and modes of action of sRNAs from horizontally acquired genomic regions remain little understood. The IsrK sRNA of Gifsy-1 prophage of Salmonella belongs to the latter class. This regulatory RNA exists in two isoforms. The first forms, when a portion of transcripts originating from isrK promoter reads-through the IsrK transcription-terminator producing a translationally inactive mRNA target. Acting in trans, the second isoform, short IsrK RNA, binds the inactive transcript rendering it translationally active. By switching on translation of the first isoform, short IsrK indirectly activates the production of AntQ, an antiterminator protein located upstream of isrK. Expression of antQ globally interferes with transcription termination resulting in bacterial growth arrest and ultimately cell death. Escherichia coli and Salmonella cells expressing AntQ display condensed chromatin morphology and localization of UvrD to the nucleoid. The toxic phenotype of AntQ can be rescued by co-expression of the transcription termination factor, Rho, or RNase H, which protects genomic DNA from breaks by resolving R-loops. We propose that AntQ causes conflicts between transcription and replication machineries and thus promotes DNA damage. The isrK locus represents a unique example of an island-encoded sRNA that exerts a highly complex regulatory mechanism to tune the expression of a toxic protein

Binding of <i>isrK-orf45</i> by IsrK.

<p>(A) Target RNAs, <i>isrK</i>-<i>orf45</i> wild type and mutant were pre incubated at 37°C (lanes 1 and 6) or 70°C (lanes 2–5 and 7–10), chilled on ice and then incubated further without or with increasing amounts of IsrK at 37°C. Arrows indicate structures A and B and binding by IsrK (Complex). (B) Incubation of <i>isrK</i>-<i>orf45</i> wild type RNA with <i>isrK</i>G31A mutant results in no binding. (C) Analysis of the RNA samples on denaturing gels exhibits one form.</p

Expression of <i>antQ</i> in <i>Salmonella</i> results in genome condensation.

<p>Fluorescence microscopy images. Cultures of <i>Salmonella</i> carrying P<i>tac</i>-<i>lacI</i> or P<i>tac</i>-<i>antQ</i>-<i>lacI</i> were diluted 1/100 and let grown for 1 hour prior to the addition of 1 mM IPTG or 100 μg/ml nalidixic acid. Samples were taken at 60 min. of treatment. DNA stained blue with DAPI.</p

IsrK facilitates 30S binding to <i>orf45</i> RBS.

<p>(A) <i>In vitro</i> synthesized RNA templates were incubated with 30S ribosomes and/or IsrK RNA prior to the addition of DMS. Thereafter, the samples were treated with phenol as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005975#sec020" target="_blank">Materials and Methods</a>. The modified sites were detected by primer extension. The numbers on the right indicate sequence positions relative to the transcription start site. (B) Open circles indicate modification sites protected by 30S binding. The initiation codons of <i>orf45</i> are indicated in red.</p

A model for <i>orf45-anrP</i> translation control by IsrK expressed in <i>trans</i>.

<p>The full-length wild type RNA forms a stable translationally inactive structure A. Some of the RNA forms structure B. IsrK sRNA expressed in <i>trans</i> binds the lower strand of helix b-I (blue), releasing the upper strand (purple) to form hairpin b-II, thus shifting the equilibrium towards structure B. In structure B, IsrK binding of the lower strand of helix d-II, renders the RBS of <i>orf45</i> accessible to ribosomes. Ribosomes bound to <i>orf45</i> elongate downstream to translate <i>anrP</i>. The two initiation codons and the stop of <i>orf45</i> are marked in red (AUG and arrow). IsrK sRNA is marked in green (dashed line).</p

Alternative structures at the <i>isrK</i> locus.

<p>(A and B) The initiation codons of <i>orf45</i> and <i>anrP</i> are in red. The stop codon of <i>orf45</i> is marked by a red arrow. The helices (a to e) are marked in Magenta. In structure A, the middle part of IsrK (in purple) is engaged in helix b-I that is complementary to the 3’ end of <i>orf45</i> (in blue). In structure B, the purple sequence forms the middle hairpin of IsrK (b-II), whereas the <i>orf45</i> blue sequence is part of helix d-II. (C) Native gel analysis. (Top panel) <i>In vitro</i> synthesized <i>isrK-orf45</i> RNAs wild type and mutants as indicated were separated on non-denaturing polyacrylamide gels. The RNAs were detected using end-labeled primer complementary to sequences of <i>orf45</i>. Arrows indicate the two conformations observed. To verify the integrity of the RNAs, the samples were analyzed on denaturing 6% polyacrylamide and 7.8 M Urea gel (Bottom panel).</p