Mohammad Ali Faghihi

Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of β-secretase

Recent efforts have revealed that numerous protein-coding messenger RNAs have natural antisense transcript partners, most of which seem to be noncoding RNAs. Here we identify a conserved noncoding antisense transcript for β-secretase-1 (BACE1), a crucial enzyme in Alzheimers disease pathophysiology. The BACE1-antisense transcript (BACE1-AS) regulates BACE1 mRNA and subsequently BACE1 protein expression in vitro and in vivo. Upon exposure to various cell stressors including amyloid-β 1–42 (Aβ 1–42), expression of BACE1-AS becomes elevated, increasing BACE1 mRNA stability and generating additional Aβ 1–42 through a post-transcriptional feed-forward mechanism. BACE1-AS concentrations were elevated in subjects with Alzheimers disease and in amyloid precursor protein transgenic mice. These data show that BACE1 mRNA expression is under the control of a regulatory noncoding RNA that may drive Alzheimers disease–associated pathophysiology. In summary, we report that a long noncoding RNA is directly implicated in the increased abundance of Aβ 1–42 in Alzheimers disease.

Regulatory roles of natural antisense transcripts

Mammalian genomes encode numerous natural antisense transcripts, but the function of these transcripts is not well understood. Functional validation studies indicate that antisense transcripts are not a uniform group of regulatory RNAs but instead belong to multiple categories with some common features. Recent evidence indicates that antisense transcripts are frequently functional and use diverse transcriptional and post-transcriptional gene regulatory mechanisms to carry out a wide variety of biological roles.

Inhibition of natural antisense transcripts in vivo results in gene-specific transcriptional upregulation

The ability to specifically upregulate genes in vivo holds great therapeutic promise. Here we show that inhibition or degradation of natural antisense transcripts (NATs) by single-stranded oligonucleotides or siRNAs can transiently and reversibly upregulate locus-specific gene expression. Brain-derived neurotrophic factor (BDNF) is normally repressed by a conserved noncoding antisense RNA transcript, BDNF-AS. Inhibition of this transcript upregulates BDNF mRNA by two- to sevenfold, alters chromatin marks at the BDNF locus, leads to increased protein levels and induces neuronal outgrowth and differentiation both in vitro and in vivo. We also show that inhibition of NATs leads to increases in glial-derived neurotrophic factor (GDNF) and ephrin receptor B2 (EPHB2) mRNA. Our data suggest that pharmacological approaches targeting NATs can confer locus-specific gene upregulation effects.Here we demonstrate that natural antisense transcripts (NATs), which are abundant in mammalian genomes, can function as repressors of specific genomic loci and that their removal or inhibition by AntagoNAT oligonucleotides leads to transient and reversible upregulation of sense gene expression. As one example, we show that Brain-Derived Neurotrophic Factor (BDNF) is under the control of a conserved noncoding antisense RNA transcript, BDNF-AS, both in vitro and in vivo. BDNF-AS tonically represses BDNF sense RNA transcription by altering chromatin structure at the BDNF locus, which in turn reduces endogenous BDNF protein and function. By providing additional and analogous examples of endogenous mRNA upregulation, we suggest that antisense RNA mediated transcriptional suppression is a common phenomenon. In sum, we demonstrate a novel pharmacological strategy by which endogenous gene expression can be upregulated in a locus-specific manner.

Evidence for natural antisense transcript-mediated inhibition of microRNA function

BackgroundMicroRNAs (miRNAs) have the potential to regulate diverse sets of mRNA targets. In addition, mammalian genomes contain numerous natural antisense transcripts, most of which appear to be non-protein-coding RNAs (ncRNAs). We have recently identified and characterized a highly conserved non-coding antisense transcript for beta-secretase-1 (BACE1), a critical enzyme in Alzheimers disease pathophysiology. The BACE1-antisense transcript is markedly up-regulated in brain samples from Alzheimers disease patients and promotes the stability of the (sense) BACE1 transcript.ResultsWe report here that BACE1-antisense prevents miRNA-induced repression of BACE1 mRNA by masking the binding site for miR-485-5p. Indeed, miR-485-5p and BACE1-antisense compete for binding within the same region in the open reading frame of the BACE1 mRNA. We observed opposing effects of BACE1-antisense and miR-485-5p on BACE1 protein in vitro and showed that Locked Nucleic Acid-antimiR mediated knockdown of miR-485-5p as well as BACE1-antisense over-expression can prevent the miRNA-induced BACE1 suppression. We found that the expression of BACE1-antisense as well as miR-485-5p are dysregulated in RNA samples from Alzheimers disease subjects compared to control individuals.ConclusionsOur data demonstrate an interface between two distinct groups of regulatory RNAs in the computation of BACE1 gene expression. Moreover, bioinformatics analyses revealed a theoretical basis for many other potential interactions between natural antisense transcripts and miRNAs at the binding sites of the latter.

MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction

N-methyl-d-aspartate (NMDA) glutamate receptors are regulators of fast neurotransmission and synaptic plasticity in the brain. Disruption of NMDA-mediated glutamate signaling has been linked to behavioral deficits displayed in psychiatric disorders such as schizophrenia. Recently, noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators of neuronal functions. Here we show that pharmacological (dizocilpine) or genetic (NR1 hypomorphism) disruption of NMDA receptor signaling reduces levels of a brain-specific miRNA, miR-219, in the prefrontal cortex (PFC) of mice. Consistent with a role for miR-219 in NMDA receptor signaling, we identify calcium/calmodulin-dependent protein kinase II γ subunit (CaMKIIγ), a component of the NMDA receptor signaling cascade, as a target of miR-219. In vivo inhibition of miR-219 by specific antimiR in the murine brain significantly modulated behavioral responses associated with disrupted NMDA receptor transmission. Furthermore, pretreatment with the antipsychotic drugs haloperidol and clozapine prevented dizocilpine-induced effects on miR-219. Taken together, these data support an integral role for miR-219 in the expression of behavioral aberrations associated with NMDA receptor hypofunction.

Regulation of chromatin structure by long noncoding RNAs: focus on natural antisense transcripts

In the decade following the publication of the Human Genome, noncoding RNAs (ncRNAs) have reshaped our understanding of the broad landscape of genome regulation. During this period, natural antisense transcripts (NATs), which are transcribed from the opposite strand of either protein or non-protein coding genes, have vaulted to prominence. Recent findings have shown that NATs can exert their regulatory functions by acting as epigenetic regulators of gene expression and chromatin remodeling. Here, we review recent work on the mechanisms of epigenetic modifications by NATs and their emerging role as master regulators of chromatin states. Unlike other long ncRNAs, antisense RNAs usually regulate their counterpart sense mRNA in cis by bridging epigenetic effectors and regulatory complexes at specific genomic loci. Understanding the broad range of effects of NATs will shed light on the complex mechanisms that regulate chromatin remodeling and gene expression in development and disease.

A small molecule enhances RNA interference and promotes microRNA processing

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are sequence-specific post-transcriptional regulators of gene expression. Although major components of the RNA interference (RNAi) pathway have been identified, regulatory mechanisms for this pathway remain largely unknown. Here we demonstrate that the RNAi pathway can be modulated intracellularly by small molecules. We have developed a cell-based assay to monitor the activity of the RNAi pathway and find that the small-molecule enoxacin (Penetrex) enhances siRNA-mediated mRNA degradation and promotes the biogenesis of endogenous miRNAs. We show that this RNAi-enhancing activity depends on the trans-activation-responsive region RNA-binding protein. Our results provide a proof-of-principle demonstration that small molecules can be used to modulate the activity of the RNAi pathway. RNAi enhancers may be useful in the development of research tools and therapeutics.

A novel RNA transcript with antiapoptotic function is silenced in fragile X syndrome.

Several genome-wide transcriptomics efforts have shown that a large percentage of the mammalian genome is transcribed into RNAs, however, only a small percentage (1–2%) of these RNAs is translated into proteins. Currently there is an intense interest in characterizing the function of the different classes of noncoding RNAs and their relevance to human disease. Using genomic approaches we discovered FMR4, a primate-specific noncoding RNA transcript (2.4 kb) that resides upstream and likely shares a bidirectional promoter with FMR1. FMR4 is a product of RNA polymerase II and has a similar half-life to FMR1. The CGG expansion in the 5′ UTR of FMR1 appears to affect transcription in both directions as we found FMR4, similar to FMR1, to be silenced in fragile X patients and up-regulated in premutation carriers. Knockdown of FMR4 by several siRNAs did not affect FMR1 expression, nor vice versa, suggesting that FMR4 is not a direct regulatory transcript for FMR1. However, FMR4 markedly affected human cell proliferation in vitro; siRNAs knockdown of FMR4 resulted in alterations in the cell cycle and increased apoptosis, while the overexpression of FMR4 caused an increase in cell proliferation. Collectively, our results demonstrate an antiapoptotic function of FMR4 and provide evidence that a well-studied genomic locus can show unexpected functional complexity. It cannot be excluded that altered FMR4 expression might contribute to aspects of the clinical presentation of fragile X syndrome and/or related disorders.

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

BackgroundAntisense 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.ResultsWe 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.ConclusionCollectively, 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 human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

BackgroundMutations in the PTEN induced putative kinase 1 (PINK1) are implicated in early-onset Parkinsons disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila.ResultsHerein we characterize a novel splice variant of PINK1 (svPINK1) that is homologous to the C-terminus regulatory domain of the protein kinase. Naturally occurring non-coding antisense provides sophisticated mechanisms for diversifying genomes and we describe a human specific non-coding antisense expressed at the PINK1 locus (naPINK1). We further demonstrate that PINK1 varies in vivo when human skeletal muscle mitochondrial content is enhanced, supporting the idea that PINK1 has a physiological role in mitochondrion. The observation of concordant regulation of svPINK1 and naPINK1 during in vivo mitochondrial biogenesis was confirmed using RNAi, where selective targeting of naPINK1 results in loss of the PINK1 splice variant in neuronal cell lines.ConclusionOur data presents the first direct observation that a mammalian non-coding antisense molecule can positively influence the abundance of a cis-transcribed mRNA under physiological abundance conditions. While our analysis implies a possible human specific and dsRNA-mediated mechanism for stabilizing the expression of svPINK1, it also points to a broader genomic strategy for regulating a human disease locus and increases the complexity through which alterations in the regulation of the PINK1 locus could occur.