Small RNAs as regulators of primary and secondary metabolism in Pseudomonas species.

Small RNAs (sRNAs) exert important functions in pseudomonads. Classical sRNAs comprise the 4.5S, 6S, 10Sa and 10Sb RNAs, which are known in enteric bacteria as part of the signal recognition particle, a regulatory component of RNA polymerase, transfer-messenger RNA (tmRNA) and the RNA component of RNase P, respectively. Their homologues in pseudomonads are presumed to have analogous functions. Other sRNAs of pseudomonads generally have little or no sequence similarity with sRNAs of enteric bacteria. Numerous sRNAs repress or activate the translation of target mRNAs by a base-pairing mechanism. Examples of this group in Pseudomonas aeruginosa are the iron-repressible PrrF1 and PrrF2 sRNAs, which repress the translation of genes encoding iron-containing proteins, and PhrS, an anaerobically inducible sRNA, which activates the expression of PqsR, a regulator of the Pseudomonas quinolone signal. Other sRNAs sequester RNA-binding proteins that act as translational repressors. Examples of this group in P. aeruginosa include RsmY and RsmZ, which are central regulatory elements in the GacS/GacA signal transduction pathway, and CrcZ, which is a key regulator in the CbrA/CbrB signal transduction pathway. These pathways largely control the extracellular activities (including virulence traits) and the selection of the energetically most favourable carbon sources, respectively, in pseudomonads.

Cloning and molecular characterization of Trypanosoma cruzi U2, U4, U5, and U6 small nuclear RNAs

Small nuclear RNAs (snRNAs) are important factors in the functioning of eukaryotic cells that form several small complexes with proteins; these ribonucleoprotein particles (U snRNPs) have an essential role in the pre-mRNA processing, particularly in splicing, catalyzed by spliceosomes, large RNA-protein complexes composed of various snRNPs. Even though they are well defined in mammals, snRNPs are still not totally characterized in certain trypanosomatids as Trypanosoma cruzi. For this reason we subjected snRNAs (U2, U4, U5, and U6) from T. cruzi epimastigotes to molecular characterization by polymerase chain reaction (PCR) and reverse transcription-PCR. These amplified sequences were cloned, sequenced, and compared with those other of trypanosomatids. Among these snRNAs, U5 was less conserved and U6 the most conserved. Their respective secondary structures were predicted and compared with known T. brucei structures. In addition, the copy number of each snRNA in the T. cruzi genome was characterized by Southern blotting.

Cl gene cluster encoding several small nucleolar RNAs: a comparison amongst trypanosomatids

Small nucleolar RNAs (snoRNAs) are small non-coding RNAs that modify RNA molecules such as rRNA and snRNA by guiding 2'-O-ribose methylation (C/D box snoRNA family) and pseudouridylation reactions (H/ACA snoRNA family). H/ACA snoRNAs are also involved in trans-splicing in trypanosomatids. The aims of this work were to characterise the Cl gene cluster that encodes several snoRNAs in Trypanosoma rangeli and compare it with clusters from Trypanosoma cruzi, Trypanosoma brucei, Leishmania major, Leishmania infantum, Leishmania braziliensis and Leptomonas collosoma. The T. rangeli Cl gene cluster is an 801 base pair (bp) repeat sequence that encodes three C/D (Cl1, Cl2 and Cl4) and three H/ACA (Cl3, Cl5 and Cl6) snoRNAs. In contrast to T. brucei, the Cl3 and Cl5 homologues have not been annotated in the Leishmania or T. cruzi genome projects (http//:www.genedb.org). Of note, snoRNA transcribed regions have a high degree of sequence identity among all species and share gene synteny. Collectively, these findings suggest that the Cl cluster could constitute an interesting target for therapeutic (gene silencing) or diagnostic intervention strategies (PCR-derived tools).

Azithromycin inhibits expression of the GacA-dependent small RNAs RsmY and RsmZ in Pseudomonas aeruginosa.

Azithromycin at clinically relevant doses does not inhibit planktonic growth of the opportunistic pathogen Pseudomonas aeruginosa but causes markedly reduced formation of biofilms and quorum-sensing-regulated extracellular virulence factors. In the Gac/Rsm signal transduction pathway, which acts upstream of the quorum-sensing machinery in P. aeruginosa, the GacA-dependent untranslated small RNAs RsmY and RsmZ are key regulatory elements. As azithromycin treatment and mutational inactivation of gacA have strikingly similar phenotypic consequences, the effect of azithromycin on rsmY and rsmZ expression was investigated. In planktonically growing cells, the antibiotic strongly inhibited the expression of both small RNA genes but did not affect the expression of the housekeeping gene proC. The azithromycin treatment resulted in reduced expression of gacA and rsmA, which are known positive regulators of rsmY and rsmZ, and of the PA0588-PA0584 gene cluster, which was discovered as a novel positive regulatory element involved in rsmY and rsmZ expression. Deletion of this cluster resulted in diminished ability of P. aeruginosa to produce pyocyanin and to swarm. The results of this study indicate that azithromycin inhibits rsmY and rsmZ transcription indirectly by lowering the expression of positive regulators of these small RNA genes.

Nucleotide sequence of the 5' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5' splicing site for subgenomic mRNAs.

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).

Small and long non-coding RNAs in cardiac homeostasis and regeneration

Cardiovascular diseases and in particular heart failure are major causes of morbidity and mortality in the Western world. Recently, the notion of promoting cardiac regeneration as a means to replace lost cardiomyocytes in the damaged heart has engendered considerable research interest. These studies envisage the utilization of both endogenous and exogenous cellular populations, which undergo highly specialized cell fate transitions to promote cardiomyocyte replenishment. Such transitions are under the control of regenerative gene regulatory networks, which are enacted by the integrated execution of specific transcriptional programs. In this context, it is emerging that the non-coding portion of the genome is dynamically transcribed generating thousands of regulatory small and long non-coding RNAs, which are central orchestrators of these networks. In this review, we discuss more particularly the biological roles of two classes of regulatory non-coding RNAs, i.e. microRNAs and long non-coding RNAs, with a particular emphasis on their known and putative roles in cardiac homeostasis and regeneration. Indeed, manipulating non-coding RNA-mediated regulatory networks could provide keys to unlock the dormant potential of the mammalian heart to regenerate. This should ultimately improve the effectiveness of current regenerative strategies and discover new avenues for repair. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens.

The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the -10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.

Small RNAs in bacterialcell-cell communication

When all three separate quorum-sensing signals act in concert in Vibrio harveyi, they maximize bioluminescence and fully repress type III secretion. V. harveyi has five qrr loci encoding small RNA regulatory molecules, each consisting of about 100 nucleotides; several of them are involved in repressing bioluminescence. Small RNAs also play roles in population density-dependent activities, including regulation of virulence factors, for bacterial pathogens such as Pseudomonas fluorescens, V. cholerae, Salmonella enterica, Pseudomonas aeruginosa, and Erwinia spp. Although some bacteria appear to carry redundant copies of small RNA genes with which to finely tune expression

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acyl-homoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens.

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

Common noncoding UMOD gene variants induce salt-sensitive hypertension and kidney damage by increasing uromodulin expression.

Hypertension and chronic kidney disease (CKD) are complex traits representing major global health problems. Multiple genome-wide association studies have identified common variants in the promoter of the UMOD gene, which encodes uromodulin, the major protein secreted in normal urine, that cause independent susceptibility to CKD and hypertension. Despite compelling genetic evidence for the association between UMOD risk variants and disease susceptibility in the general population, the underlying biological mechanism is not understood. Here, we demonstrate that UMOD risk variants increased UMOD expression in vitro and in vivo. Uromodulin overexpression in transgenic mice led to salt-sensitive hypertension and to the presence of age-dependent renal lesions similar to those observed in elderly individuals homozygous for UMOD promoter risk variants. The link between uromodulin and hypertension is due to activation of the renal sodium cotransporter NKCC2. We demonstrated the relevance of this mechanism in humans by showing that pharmacological inhibition of NKCC2 was more effective in lowering blood pressure in hypertensive patients who are homozygous for UMOD promoter risk variants than in other hypertensive patients. Our findings link genetic susceptibility to hypertension and CKD to the level of uromodulin expression and uromodulin's effect on salt reabsorption in the kidney. These findings point to uromodulin as a therapeutic target for lowering blood pressure and preserving renal function.

A pathogenic mechanism in Huntington"s disease involves small CAG-repeated RNAs with neurotoxic activity

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches

Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

AIM: Heart disease is recognized as a consequence of dysregulation of cardiac gene regulatory networks. Previously, unappreciated components of such networks are the long non-coding RNAs (lncRNAs). Their roles in the heart remain to be elucidated. Thus, this study aimed to systematically characterize the cardiac long non-coding transcriptome post-myocardial infarction and to elucidate their potential roles in cardiac homoeostasis. METHODS AND RESULTS: We annotated the mouse transcriptome after myocardial infarction via RNA sequencing and ab initio transcript reconstruction, and integrated genome-wide approaches to associate specific lncRNAs with developmental processes and physiological parameters. Expression of specific lncRNAs strongly correlated with defined parameters of cardiac dimensions and function. Using chromatin maps to infer lncRNA function, we identified many with potential roles in cardiogenesis and pathological remodelling. The vast majority was associated with active cardiac-specific enhancers. Importantly, oligonucleotide-mediated knockdown implicated novel lncRNAs in controlling expression of key regulatory proteins involved in cardiogenesis. Finally, we identified hundreds of human orthologues and demonstrate that particular candidates were differentially modulated in human heart disease. CONCLUSION: These findings reveal hundreds of novel heart-specific lncRNAs with unique regulatory and functional characteristics relevant to maladaptive remodelling, cardiac function and possibly cardiac regeneration. This new class of molecules represents potential therapeutic targets for cardiac disease. Furthermore, their exquisite correlation with cardiac physiology renders them attractive candidate biomarkers to be used in the clinic.