Reexamining assumptions about miRNA-guided gene silencing.

MicroRNAs (miRNAs) are short endogenously expressed RNAs that have the potential to regulate the expression of any RNA. This potential has led to the publication of several thousand papers each year connecting miRNAs to many different genes and human diseases. By contrast, relatively few papers appear that investigate the molecular mechanism used by miRNAs. There is a disconnect between rigorous understanding of mechanism and the extraordinary diversity of reported roles for miRNAs. Consequences of this disconnect include confusion about the assumptions underlying the basic science of human miRNAs and slow development of therapeutics that target miRNAs. Here, we present an overview of investigations into miRNAs and their impact on gene expression. Progress in our understanding of miRNAs would be aided by a greater focus on the mechanism of miRNAs and a higher burden of evidence on researchers who seek to link expression of a particular miRNA to a biological phenotype.

Progress towards drug discovery for Friedreich's Ataxia: Identifying synthetic oligonucleotides that more potently activate expression of human frataxin protein.

Friedreich's Ataxia (FRDA) is an incurable genetic disease caused by an expanded trinucleotide AAG repeat within intronic RNA of the frataxin (FXN) gene. We have previously demonstrated that synthetic antisense oligonucleotides or duplex RNAs that are complementary to the expanded repeat can activate expression of FXN and return levels of FXN protein to near normal. The potency of these compounds, however, was too low to encourage vigorous pre-clinical development. We now report testing of "gapmer" oligonucleotides consisting of a central DNA portion flanked by chemically modified RNA that increases binding affinity. We find that gapmer antisense oligonucleotides are several fold more potent activators of FXN expression relative to previously tested compounds. The potency of FXN activation is similar to a potent benchmark gapmer targeting the nuclear noncoding RNA MALAT-1, suggesting that our approach has potential for developing more effective compounds to regulate FXN expression in vivo.

Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

Activating frataxin expression by repeat-targeted nucleic acids.

Friedreich's ataxia is an incurable genetic disorder caused by a mutant expansion of the trinucleotide GAA within an intronic FXN RNA. This expansion leads to reduced expression of frataxin (FXN) protein and evidence suggests that transcriptional repression is caused by an R-loop that forms between the expanded repeat RNA and complementary genomic DNA. Synthetic agents that increase levels of FXN protein might alleviate the disease. We demonstrate that introducing anti-GAA duplex RNAs or single-stranded locked nucleic acids into patient-derived cells increases FXN protein expression to levels similar to analogous wild-type cells. Our data are significant because synthetic nucleic acids that target GAA repeats can be lead compounds for restoring curative FXN levels. More broadly, our results demonstrate that interfering with R-loop formation can trigger gene activation and reveal a new strategy for upregulating gene expression.

Promoter RNA links transcriptional regulation of inflammatory pathway genes.

Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.

Antisense transcripts are targets for activating small RNAs.

Agents that activate expression of specific genes to probe cellular pathways or alleviate disease would go beyond existing approaches for controlling gene expression. Duplex RNAs complementary to promoter regions can repress or activate gene expression. The mechanism of these promoter-directed antigene RNAs (agRNAs) has been obscure. Other work has revealed noncoding transcripts that overlap mRNAs. The function of these noncoding transcripts is also not understood. Here we link these two sets of enigmatic results. We find that antisense transcripts are the target for agRNAs that activate or repress expression of progesterone receptor (PR). agRNAs recruit Argonaute proteins to PR antisense transcripts and shift localization of the heterogeneous nuclear ribonucleoprotein-k, RNA polymerase II and heterochromatin protein 1 gamma. Our data demonstrate that antisense transcripts have a central role in recognition of the PR promoter by both activating and inhibitory agRNAs.

Inhibiting gene expression with peptide nucleic acid (PNA)--peptide conjugates that target chromosomal DNA.

Peptide nucleic acids (PNAs) are nonionic DNA/RNA mimics that can recognize complementary sequences by Watson-Crick base pairing. The neutral PNA backbone facilitates the recognition of duplex DNA by strand invasion, suggesting that antigene PNAs (agPNAs) can be important tools for exploring the structure and function of chromosomal DNA inside cells. However, before agPNAs can enter wide use, it will be necessary to develop straightforward strategies for introducing them into cells. Here, we demonstrate that agPNA-peptide conjugates can target promoter DNA and block progesterone receptor (PR) gene expression inside cells. Thirty-six agPNA-peptide conjugates were synthesized and tested. We observed inhibition of gene expression using cationic peptides containing either arginine or lysine residues, with eight or more cationic amino acids being preferred. Both 13 and 19 base agPNA-peptide conjugates were inhibitory. Inhibition was observed in human cancer cell lines expressing either high or low levels of progesterone receptor. Modification of agPNA-peptide conjugates with hydrophobic amino acids or small molecule hydrophobic moieties yielded improved potency. Inhibition by agPNAs did not require cationic lipid or any other additive, but adding agents to cell growth media that promote endosomal release caused modest increases in agPNA potency. These data demonstrate that chromosomal DNA is accessible to agPNA-peptide conjugates and that chemical modifications can improve potency.

The synaptic complex of RecA protein participates in hybridization and inverse strand exchange reactions.

RecA protein catalyzes strand exchange between homologous single-stranded and double-stranded DNAs. In the presence of ATPgammaS, the post-strand exchange synaptic complex is a stable end product that can be studied. Here we ask whether such complexes can hybridize to or exchange with DNA, 2'-OMe RNA, PNA, or LNA oligonucleotides. Using a gel mobility shift assay, we show that the displaced strand of a 45 bp synaptic complex can hybridize to complementary oligonucleotides with different backbones to form a four-stranded (double D-loop) joint that survives removal of the RecA protein. This hybridization reaction, which confirms the single-stranded character of the displaced strand in a synaptic complex, might initiate recombination-dependent DNA replication if it occurs in vivo. We also show that either strand of the heteroduplex in a 30 bp synaptic complex can be replaced with a homologous DNA oligonucleotide in a strand exchange reaction that is mediated by the RecA filament. Consistent with the important role that deoxyribose plays in strand exchange, oligonucleotides with non-DNA backbones did not participate in this reaction. The hybridization and strand exchange reactions reported here demonstrate that short synaptic complexes are dynamic structures even in the presence of ATPgammaS.