Transcriptional Silencing by Single-Stranded RNAs Targeting a Noncoding RNA That Overlaps a Gene Promoter

Departments of Pharmacology and Biochemistry, UT Southwestern Medical Center , 6001 Forest Park Road, Dallas, Texas 75390-9041, United States.
ACS Chemical Biology (Impact Factor: 5.33). 10/2012; 8(1). DOI: 10.1021/cb300490j
Source: PubMed


RNAi using single-strand RNA would provide new options for therapeutic development and for investigating critical questions of mechanism. Using chemically modified single-strands, we test the hypothesis that single-stranded RNAs can engage the RNAi pathway and silence gene transcription. We find that a chemically modified single-stranded silencing RNA (ss-siRNA) designed to be complementary to a long noncoding RNA (lncRNA) requires argonaute protein, functions through the RNAi pathway, and inhibits gene transcription. These data expand the use of single-stranded RNA to cell nuclei.

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Available from: David Reid Corey, Aug 27, 2015
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    • "The relevance of Csmd1 for brain functions was further emphasized by the identification of a Csmd1 promoter-associated lncRNA, possibly responsible for brain-specific promoter activity in the adult and developing CNS (Figure 3). Such promoter-associated lncRNAs is a new class of long untranslated RNA which may underlie a functional coupling of transcription and translation/splicing [25], [26]. Finally, we also demonstrate that depletion of Csmd1 has a remarkably limited effect on the whole transcriptome (Figure 2). "
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    ABSTRACT: Recent meta-analyses of schizophrenia genome-wide association studies (GWASs) have identified the CUB and SUSHI multiple domains 1 (CSMD1) gene as a statistically strong risk factor. CSMD1 is a complement control-related protein suggested to inhibit the classical complement pathway, being expressed in developing neurons. However, expression of CSMD1 is largely uncharacterized and relevance for behavioral phenotypes is not previously demonstrated. Here, we assess neuropsychological behaviors of a Csmd1 knockout (KO) mouse in a selection of standard behavioral tests. Deregulation of neuropsychological responses were observed in both the open field and the elevated plus maze tests, in which KO mice spent 55% and 33% less time than WT littermate mice in open areas, respectively. Altered behaviors were also observed in tail suspension and to higher acoustic stimuli, for which Csmd1 KO mice showed helplessness and moderate increase in startle amplitude, respectively. Furthermore, Csmd1 KO mice also displayed increased weight-gain and glucose tolerance, similar to a major phenotype of the metabolic syndrome that also has been associated to the human CSMD1 locus. Consistent with a role in the control of behaviors, Csmd1 was found highly expressed in the central nervous system (CNS), and with some expression in visceral fat and ovary, under tissue-specific control by a novel promoter-associated lncRNA. In summary, disruption of Csmd1 induces behaviors reminiscent of blunted emotional responses, anxiety and depression. These observations suggest an influence of the CSMD1 schizophrenia susceptibility gene on psychopathology and endophenotypes of the negative symptom spectra.
    PLoS ONE 11/2013; 8(11):e79501. DOI:10.1371/journal.pone.0079501 · 3.23 Impact Factor
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    • "Although the mechanisms of RNA-induced transcriptional regulation is largely unclear, recent studies suggested that non-coding (nc) RNAs activate transcription by removing repressive entities or promoting activating epigenetic marks. It was shown that promoter-overlapping ncRNA transcripts that usually repress transcription of the locus are targets of antigene RNAs (agRNAs), which either degrade the ncRNA transcripts to activate transcription or form an intact transcript-Ago-agRNA complex that acts as a scaffold to recruit other proteins and RNA Polymerase II at the target site to establish activating chromatin marks and induce transcription [9], [22], [23], [24]. We observed that miR-324-3p targets the RelA promoter in close proximity (within 100 bp) of the transcription start site (TSS) suggesting that the Ago2-miR-324-3p complex might recruit other proteins such as EZHs to open chromatin and activate transcription at the TSS. "
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    ABSTRACT: MicroRNAs (miRNAs) are known to repress translation by binding to the 3'UTRs of mRNAs. Using bioinformatics, we recently reported that several miRNAs also have target sites in DNA particularly in the promoters of the protein-coding genes. To understand the functional significance of this phenomenon, we tested the effects of miR-324-3p binding to RelA promoter. In PC12 cells, co-transfection with premiR-324-3p induced a RelA promoter plasmid in a dose-dependent manner and this effect was lost when the miR-324-3p binding site in the promoter was mutated. PremiR-324-3p transfection also significantly induced the endogenous RelA mRNA and protein expression in PC12 cells. Furthermore, transfection with premiR-324-3p increased the levels of cleaved caspase-3 which is a marker of apoptosis. Importantly, the miR-324-3p effects were Ago2 mediated as Ago2 knockdown prevented RelA expression and cleavage of caspase-3. Thus, our studies show that miRNA-mediated transcriptional activation can be seen in PC12 cells which are neural in origin.
    PLoS ONE 11/2013; 8(11):e79467. DOI:10.1371/journal.pone.0079467 · 3.23 Impact Factor
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    • "Instead of the slicer mechanism used by AGO2, when RNAs were fully complementary to their targets (25,26), the imperfect base-pairing permits binding by AGO2 but not cleavage of the target mRNA. These introduced mismatches led to improved selectivity for duplex RNAs and ss-siRNAs (12,15,24,27,28). "
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    ABSTRACT: Single-stranded silencing RNAs (ss-siRNAs) provide an alternative approach to gene silencing. ss-siRNAs combine the simplicity and favorable biodistribution of antisense oligonucleotides with robust silencing through RNA interference (RNAi). Previous studies reported potent and allele-selective inhibition of human huntingtin expression by ss-siRNAs that target the expanded CAG repeats within the mutant allele. Mutant ataxin-3, the genetic cause of Machado-Joseph Disease, also contains an expanded CAG repeat. We demonstrate here that ss-siRNAs are allele-selective inhibitors of ataxin-3 expression and then redesign ss-siRNAs to optimize their selectivity. We find that both RNAi-related and non-RNAi-related mechanisms affect gene expression by either blocking translation or affecting alternative splicing. These results have four broad implications: (i) ss-siRNAs will not always behave similarly to analogous RNA duplexes; (ii) the sequences surrounding CAG repeats affect allele-selectivity of anti-CAG oligonucleotides; (iii) ss-siRNAs can function through multiple mechanisms and; and (iv) it is possible to use chemical modification to optimize ss-siRNA properties and improve their potential for drug discovery.
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