Emerging similarities in epigenetic gene silencing by long noncoding RNAs

Long noncoding RNAs (lncRNAs) such as Xist, Air, and Kcnq1ot1 are required for epigenetic silencing of multiple genes in cis within large chromosomal domains, including distant genes located hundreds of kilobase pairs away. Recent evidence suggests that all three of these lncRNAs are functional and that they silence gene expression, in part, through an intimate interaction with chromatin. Here we provide an overview of lncRNA-dependent gene silencing, focusing on recent findings for the Air and Kcnq1ot1 lncRNAs. We review molecular evidence indicating that these lncRNAs interact with chromatin and correlate their presence with specific histone modifications associated with gene silencing. A general model for a lncRNA-dependent gene-silencing mechanism is presented based on the apparent ability of lncRNAs to recruit histone-modifying activities to chromatin. However, alternate mechanisms may be required to explain the silencing of some lncRNA-dependent genes. Finally, we discuss unanswered questions and future perspectives associated with these enigmatic lncRNA molecules.     

Regulation of genes encoding subunits of the trehalose synthase complex inSaccharomyces cerevisiae: novel variations of STRE-mediated transcription control?

Saccharomyces cerevisiae cells show under suboptimal growth conditions a complex response that leads to the acquisition of tolerance to different types of environmental stress. This response is characterised by enhanced expression of a number of genes which contain so-called stress-responsive elements (STREs) in their promoters. In addition, the cells accumulate under suboptimal conditions the putative stress protectant trehalose. In this work, we have examined the expression of four genes encoding subunits of the trehalose synthase complex,GGS1/TPS1, TPS2, TPS3 andTSL1. We show that expression of these genes is coregulated under stress conditions. Like for many other genes containing STREs, expression of the trehalose synthase genes is also induced by heat and osmotic stress and by nutrient starvation, and negatively regulated by the Ras-cAMP pathway. However, during fermentative growth onlyTSL1 shows an expression pattern like that of the STRE-controlled genesCTT1 andSSA3, while expression of the three other trehalose synthase genes is only transiently down-regulated. This difference in expression might be related to the known requirement of trehalose biosynthesis for the control of yeast glycolysis and hence for fermentative growth. We conclude that the mere presence in the promoter of (an) active STRE(s) does not necessarily imply complete coregulation of expression. Additional mechanisms appear to fine tune the activity of STREs in order to adapt the expression of the downstream genes to specific requirements.     

Efficient delivery of RNA Interference to peripheral neurons in vivo using herpes simplex virus

Considerable interest has been focused on inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although small interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge limiting its applications. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) using replication-defective herpes simplex viral (HSV-1) vectors. HSV-mediated delivery of short-hairpin RNA (shRNA) targeting reporter genes resulted in highly effective and specific silencing in neuronal and non-neuronal cells in culture and in the DRG of mice in vivo including in a transgenic mouse model. We further establish proof of concept by demonstrating in vivo silencing of the endogenous trpv1 gene. These data are the first to show silencing in DRG neurons in vivo by vector-mediated delivery of shRNA. Our results support the utility of HSV vectors for gene silencing in peripheral neurons and the potential application of this technology to the study of nociceptive processes and in pain gene target validation studies.     

Integrative transcriptome analysis identifies new oxidosqualene cyclase genes involved in ginsenoside biosynthesis in Jilin ginseng

BackgroundJilin ginseng, Panax ginseng, is a valuable medicinal herb whose ginsenosides are its major bioactive components. The ginseng oxidosqualene cyclase (PgOSC) gene family is known to play important roles in ginsenoside biosynthesis, but few members of the gene family have been functionally studied.MethodsThe PgOSC gene family has been studied by an integrated analysis of gene expression-ginsenoside content correlation, gene mutation-ginsenoside content association and gene co-expression network, followed by functional analysis through gene regulation.ResultsWe found that five of the genes in the PgOSC gene family, including two published ginsenoside biosynthesis genes and three new genes, were involved in ginsenoside biosynthesis. Not only were the expressions of these genes significantly correlated with ginsenoside contents, but also their nucleotide mutations significantly influenced ginsenoside contents. These results were further verified by regulation analysis of the genes by methyl jasmonate (MeJA) in ginseng hairy roots. Four of these five PgOSC genes were mapped to the ginsenoside biosynthesis pathway. These PgOSC genes expressed differently across tissues, but relatively consistent across developmental stages. These PgOSC genes formed a single co-expression network with those published ginsenoside biosynthesis genes, further confirming their roles in ginsenoside biosynthesis. When the network varied, ginsenoside biosynthesis was significantly influenced, thus revealing the molecular mechanism of ginsenoside biosynthesis.ConclusionAt least five of the PgOSC genes, including the three newly identified and two published PgOSC genes, are involved in ginsenoside biosynthesis. These results provide gene resources and knowledge essential for enhanced research and applications of ginsenoside biosynthesis in ginseng.     

Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica.     

Silencing of a second dimethylallyltryptophan synthase of <Emphasis Type="Italic">Penicillium roqueforti</Emphasis> reveals a novel clavine alkaloid gene cluster

Penicillium roqueforti produces several prenylated indole alkaloids, including roquefortine C and clavine alkaloids. The first step in the biosynthesis of roquefortine C is the prenylation of tryptophan-derived dipeptides by a dimethylallyltryptophan synthase, specific for roquefortine biosynthesis (roquefortine prenyltransferase). A second dimethylallyltryptophan synthase, DmaW2, different from the roquefortine prenyltransferase, has been studied in this article. Silencing the gene encoding this second dimethylallyltryptophan synthase, dmaW2, proved that inactivation of this gene does not prevent the production of roquefortine C, but suppresses the formation of other indole alkaloids. Mass spectrometry studies have identified these compounds as isofumigaclavine A, the pathway final product and prenylated intermediates. The silencing does not affect the production of mycophenolic acid and andrastin A. A bioinformatic study of the genome of P. roqueforti revealed that DmaW2 (renamed IfgA) is a prenyltransferase involved in isofumigaclavine A biosynthesis encoded by a gene located in a six genes cluster (cluster A). A second three genes cluster (cluster B) encodes the so-called yellow enzyme and enzymes for the late steps for the conversion of festuclavine to isofumigaclavine A. The yellow enzyme contains a tyrosine-181 at its active center, as occurs in Neosartorya fumigata, but in contrast to the Clavicipitaceae fungi. A complete isofumigaclavines A and B biosynthetic pathway is proposed based on the finding of these studies on the biosynthesis of clavine alkaloids.