RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals

BACKGROUND: Antisense transcription, yielding both coding and non-coding RNA, is a widespread phenomenon in mammals. The mechanism by which natural antisense transcripts (NAT) may regulate gene expression are largely unknown. The aim of the present study was to explore the mechanism of reciprocal sense-antisense (S-AS) regulation by studying the effects of a coding and non-coding NAT on corresponding gene expression, and to investigate the possible involvement of endogenous RNA interference (RNAi) in S-AS interactions. RESULTS: We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1α as primary examples of endogenous genes with coding and non-coding NAT partners, respectively. Here we provide direct evidence against S-AS RNA duplex formation in the cytoplasm of human cells and subsequent activation of RNAi. CONCLUSION: Collectively, our data demonstrate that NAT regulation of gene expression occurs through a pathway independent of Dicer associated RNAi. Moreover, we introduce an experimental strategy with utility for the functional examination of other S-AS pair interactions

The expression signature of in vitro senescence resembles mouse but not human aging

BACKGROUND: The biological mechanisms that underlie aging have not yet been fully identified. Senescence, a phenomenon occurring in vitro, limits the number of cell divisions in mammalian cell cultures and has been suggested to contribute to aging. RESULTS: We investigated whether the changes in gene expression that occur during mammalian aging and induction of cellular senescence are similar. We compared changes of gene expression in seven microarray datasets from aging human, mouse and rat, as well as four microarray datasets from senescent cells of man and mouse. The datasets were publicly available or obtained from other laboratories. Correlation measures were used to establish similarities of the expression profiles and gene ontology analyses to identify functional groups of genes that are co-regulated. Robust similarities were established between aging in different species and tissues, indicating that there is an aging transcriptome. Although some cross-species comparisons displayed high correlation, intra-species similarities were more reliable. Similarly, a senescence transcriptome was demonstrated that is conserved across cell types. A similarity between the expression signatures of cellular senescence and aging could be established in mouse, but not in human. CONCLUSION: Our study is the first to use microarray data from several studies and laboratories for dissection of a complex biological phenotype. We demonstrate the presence of a mammalian aging transcriptome, and discuss why similarity between cellular senescence and aging is apparent in aging mice only

Considerations when using the significance analysis of microarrays (SAM) algorithm

BACKGROUND: Users of microarray technology typically strive to use universally acceptable data analysis strategies to determine significant expression changes in their experiments. One of the most frequently utilised methods for gene expression data analysis is SAM (significance analysis of microarrays). The impact of selection thresholds, on the output from SAM, may critically alter the conclusion of a study, yet this consideration has not been systematically evaluated in any publication. RESULTS: We have examined the effect of discrete data selection criteria (qualification criteria for inclusion) and response thresholds (out-put filtering) on the number of significant genes reported by SAM. The use of a reduced data set by applying arbitrary restrictions vis-à-vis abundance calls (e.g. from D-chip) or application of the fold change (FC) option within SAM (named the FC hurdle hereafter), can substantially alter the significant gene list when running SAM in Microsoft Excel. We determined that for a given final FC criteria (e.g. 1.5 fold change) the FC hurdle applied within Microsoft Excel SAM alters the number of reported genes above the final FC criteria. The reason is that the FC hurdle changes the composition of the control data set, such that a different significance level (q-value) is obtained for any given gene. This effect can be so large that it changes subsequent post hoc analysis interpretation, such as ontology overrepresentation analysis. CONCLUSION: Our results argue for caution when using SAM. All data sets analysed with SAM could be reanalysed taking into account the potential impact of the use of arbitrary thresholds to trim data sets before significance testing

Lower rate of genomic variation identified in the trans-membrane domain of monoamine sub-class of Human G-Protein Coupled Receptors: The Human GPCR-DB Database

BACKGROUND: We have surveyed, compiled and annotated nucleotide variations in 338 human 7-transmembrane receptors (G-protein coupled receptors). In a sample of 32 chromosomes from a Nordic population, we attempted to determine the allele frequencies of 80 non-synonymous SNPs, and found 20 novel polymorphic markers. GPCR receptors of physiological and clinical importance were prioritized for statistical analysis. Natural variation and rare mutation information were merged and presented online in the Human GPCR-DB database . RESULTS: The average number of SNPs per 1000 bases of exonic sequence was found to be twice the average number of SNPs per Kilobase of intronic regions (2.2 versus 1.0). Of the 338 genes, 111 were single exon genes, that is, were intronless. The average number of exonic-SNPs per single-exon gene was 3.5 (n = 395) while that for multi-exon genes was 0.8 (n = 1176). The average number of variations within the different protein domain (N-terminus, internal- and external-loops, trans-membrane region, C-terminus) indicates a lower rate of variation in the trans-membrane region of Monoamine GPCRs, as compared to Chemokine- and Peptide-receptor sub-classes of GPCRs. CONCLUSIONS: Single-exon GPCRs on average have approximately three times the number of SNPs as compared to GPCRs with introns. Among various functional classes of GPCRs, Monoamine GPRCs have lower number of natural variations within the trans-membrane domain indicating evolutionary selection against non-synonymous changes within the membrane-localizing domain of this sub-class of GPCRs

Vector-based siRNA delivery strategies for high-throughput screening of novel target genes

Application of siRNA in high-throughput fashion is still in its early phase although the principle has been established for three years. In this review, we outline the different vector-based siRNA delivery platforms as well as resources that are becoming available for high-throughput applications, and some initial outcomes of vector siRNA high-throughput screening efforts using vector encoded siRNA. It is expected that further improvement of the siRNA technology and availability of the siRNA resources will help to materialize the potential of siRNA for functional genomics and drug target validation

Nucleic acid-based therapeutics for the treatment of central nervous system disorders

Nucleic acid-based therapeutics (NBTs) are an emerging class of drugs with potential for the treatment of a wide range of central nervous system conditions. To date, pertaining to CNS indications, there are two commercially available NBTs and a large number of ongoing clinical trials. However, these NBTs are applied directly to the brain due to very low blood brain barrier permeability. In this review, we outline recent advances in chemical modifications of NBTs and NBT delivery techniques intended to promote brain exposure, efficacy, and possible future systemic application

Pro- and Anti-inflammatory Biomarkers as Predictors of Response to Valproate in Patients with Comorbid Alcohol Use and Bipolar Disorder-Preliminary Findings

Objective/Hypothesis: Bipolar disorder (BD) has the highest association with alcohol and other substance use disorders compared to other major psychiatric disorders. This patient population is particularly challenging to treat. We have previously shown that some patients with co-occurring alcohol use and bipolar disorders respond to the GABAergic agonist valproate (VPA), which is known to modulates the dopaminergic system, and also as an epigenetic modifier. Predictors of therapeutic response to VPA in patients with AUD/BD are not known, and the subgroup which would benefit from VPA is still to be identified. Recent evidence suggests that AUD promotes a pro-inflammatory state while VPA increases levels of anti-inflammatory factors. We hypothesized that VPA has an anti-inflammatory effect and that patients with AUD/BD who respond to VPA have higher baseline inflammatory indices. Methods: Nine patients with DSM-IV-defined diagnoses of AUD and BD (AUD/BD) were enrolled in the study. Patients received a course of VPA for 3 months at an average dose of 1000 mg a day in addition to receiving either naltrexone of 50 mg daily or placebo. Blood was collected prior to the initiation of VPA and throughout the treatment study. Liver function tests and trough VPA serum concentrations were evaluated periodically. Alcohol use outcome was assessed using the Timeline Follow-Back for Recent Drinking. The use of other drugs was monitored through regular urine drug screen. The primary alcohol use outcome was changes in proportion of weekly heavy drinking days (defined as ³ 5 drinks per day for men and ³ 4 drinks per day for women). Plasma levels of cytokines were measured using Multiplex Immunoassay, in accordance to the manufacturers’ recommendations. Results: We found that about one half of enrolled patients responded to VPA. Screening of pro- and anti-inflammatory cytokines showed that responders had higher levels of the chemokine SDF-1a/CXCL12a and the pro-inflammatory marker C-reactive protein (CRP) and lower levels of anti-inflammatory factor matrix metalloproteinase-10 (MMP-10) (p \u3c 0.05). Screening of cytokines in samples before and after treatment with VPA showed that VPA increased levels of anti-inflammatory factors interleukin-10 (IL-10) and MMP-10 (p \u3c 0.05) and tended to decrease levels of pro-inflammatory CRP (p \u3e 0.05). Discussion: Pro- and anti-inflammatory biomarkers may serve as predictors of treatment response to VPA in patients with combined AUD/BD. Our preliminary results also suggest that therapeutic effect of VPA may be in part due to anti-inflammatory action of VPA. Larger studies may be indicated to validate these findings

Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2

SummaryLong non-coding RNAs (lncRNAs), including natural antisense transcripts (NATs), are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer’s disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion

Comprehensive analysis of the transcriptional landscape of the human FMR1 gene reveals two new long noncoding RNAs differentially expressed in Fragile X syndrome and Fragile X-associated tremor/ataxia syndrome

The majority of the human genome is transcribed but not translated, giving rise to noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs, >200 nt) that perform a wide range of functions in gene regulation. The Fragile X mental retardation 1 (FMR1) gene is a microsatellite locus that in the general population contains <55 CGG repeats in its 5′-untranslated region. Expansion of this repeat region to a size of 55-200 CGG repeats, known as premutation, is associated with Fragile X tremor and ataxia syndrome (FXTAS). Further expansion beyond 200 CGG repeats, or full mutation, leads to FMR1 gene silencing and results in Fragile X syndrome (FXS). Using a novel technology called “Deep-RACE”, which combines rapid amplification of cDNA ends (RACE) with next generation sequencing, we systematically interrogated the FMR1 gene locus for the occurrence of novel lncRNAs. We discovered two transcripts, FMR5 and FMR6. FMR5 is a sense lncRNA transcribed upstream of the FMR1 promoter, whereas FMR6 is an antisense transcript overlapping the 3′ region of FMR1. FMR5 was expressed in several human brain regions from unaffected individuals and from full and premutation patients. FMR6 was silenced in full mutation and, unexpectedly, in premutation carriers suggesting abnormal transcription and/or chromatin remodeling prior to transition to the full mutation. These lncRNAs may thus be useful as biomarkers, allowing for early detection and therapeutic intervention in FXS and FXTAS. Finally we show that FMR5 and FMR6 are expressed in peripheral blood leukocytes and propose future studies that correlate lncRNA expression with clinical outcomes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00439-013-1356-6) contains supplementary material, which is available to authorized users

Analysis of siRNA specificity on targets with double-nucleotide mismatches

Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that ∼35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site