Keith T. Gagnon

Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs

Many neurological disorders are caused by expanded trinucleotide repeats1, including Machado-Joseph Disease (MJD)2 and Huntington Disease (HD)3. MJD and HD are caused by expanded CAG repeats within the ataxin-3 (ATXN3) and huntingtin (HTT) genes. Inhibiting expression of ATXN3 or HTT are promising therapeutic strategies, but indiscriminant inhibition of wild-type and mutant alleles may lead to toxicity. We hypothesized that expanded triplet repeat mRNA might be preferentially recognized by complementary oligomers. We observe selective inhibition of mutant ataxin-3 and HTT protein expression by peptide nucleic acid (PNA) and locked nucleic acid (LNA) oligomers targeting CAG repeats. Duplex RNAs were less selective, suggesting an advantage for single-stranded oligomers. Inhibiting mutant HTT expression protected cultured striatal neurons from an HD mouse model against glutamate-induced toxicity. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length offer new options for treating MJD, HD, and other hereditary diseases.Expanded trinucleotide repeats cause many neurological diseases. These include Machado-Joseph disease (MJD) and Huntingtons disease (HD), which are caused by expanded CAG repeats within an allele of the ataxin-3 (ATXN3) and huntingtin (HTT) genes, respectively. Silencing expression of these genes is a promising therapeutic strategy, but indiscriminate inhibition of both the mutant and wild-type alleles may lead to toxicity, and allele-specific approaches have required polymorphisms that differ among individuals. We report that peptide nucleic acid and locked nucleic acid antisense oligomers that target CAG repeats can preferentially inhibit mutant ataxin-3 and HTT protein expression in cultured cells. Duplex RNAs were less selective than single-stranded oligomers. The activity of the peptide nucleic acids does not involve inhibition of transcription, and differences in mRNA secondary structure or the number of oligomer binding sites may be important. Antisense oligomers that discriminate between wild-type and mutant genes on the basis of repeat length may offer new options for developing treatments for MJD, HD and related hereditary diseases.

RNAi Factors Are Present and Active in Human Cell Nuclei

Summary RNAi is widely appreciated as a powerful regulator of mRNA translation in the cytoplasm of mammalian cells. However, the presence and activity of RNAi factors in the mammalian nucleus has been the subject of considerable debate. Here we show that Argonaute-2 (Ago2) and RNAi factors Dicer, TRBP and TRNC6A/GW182 are in the human nucleus and associate together in multi-protein complexes. Small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA, demonstrating that RNAi can function in the human nucleus. Nuclear Dicer is active and miRNAs are bound to nuclear Ago2, consistent with the existence of nuclear miRNA pathways. Notably, we do not detect loading of duplex small RNAs in nuclear extracts and known loading factors are absent. These results extend RNAi into the mammalian nucleus and suggest that regulation of RNAi via small RNA loading of Ago2 differs between the cytoplasm and the nucleus.

Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat

Huntingtons disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

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.

A Dimeric Structure for Archaeal Box C/D Small Ribonucleoproteins

Seeing Double A particular set of ubiquitous small (nucleolar) ribonucleoproteins are important for optimal ribosome function and protein synthesis. Bleichert et al. (p. 1384) used electron microscopy and single-particle analysis to investigate the structure of an archaeal version that contains the small RNA (sRNA) and all the associated core proteins. Unexpectedly, this ribonucleoprotein is a homodimer, formed of two sRNAs and four copies of each of the core proteins. This dimer is likely to be the enzymatically active form, as mutations disrupting di-sRNP formation inhibited activity. Electron microscopy and single-particle analysis show that a small nuclear ribonucleoprotein forms a dimeric structure. Methylation of ribosomal RNA (rRNA) is required for optimal protein synthesis. Multiple 2′-O-ribose methylations are carried out by box C/D guide ribonucleoproteins [small ribonucleoproteins (sRNPs) and small nucleolar ribonucleoproteins (snoRNPs)], which are conserved from archaea to eukaryotes. Methylation is dictated by base pairing between the specific guide RNA component of the sRNP or snoRNP and the target rRNA. We determined the structure of a reconstituted and catalytically active box C/D sRNP from the archaeon Methanocaldococcus jannaschii by single-particle electron microscopy. We found that archaeal box C/D sRNPs unexpectedly formed a dimeric structure with an alternative organization of their RNA and protein components that challenges the conventional view of their architecture. Mutational analysis demonstrated that this di-sRNP structure was relevant for the enzymatic function of archaeal box C/D sRNPs.

Argonaute and the Nuclear RNAs: New Pathways for RNA-Mediated Control of Gene Expression

Small RNAs are a commonly used tool for gene silencing and a promising platform for nucleic acid drug development. They are almost exclusively used to silence gene expression post-transcriptionally through degradation of mRNA. Small RNAs, however, can have a broader range of function by binding to Argonaute proteins and associating with complementary RNA targets in the nucleus, including long noncoding RNAs (lncRNAs) and pre-mRNA. Argonaute-RNA complexes can regulate nuclear events like transcription, genome maintenance, and splicing. Thousands of lncRNAs and alternatively spliced pre-mRNA isoforms exist in humans, and these RNAs may serve as natural targets for regulation and therapeutic intervention. This review describes nuclear mechanisms for Argonaute proteins and small RNAs, new pathways for sequence-specific targeting, and the potential for therapeutic development of small RNAs with nuclear targets.

Analysis of nuclear RNA interference in human cells by subcellular fractionation and Argonaute loading

RNAi is well known for its ability to regulate gene expression in the cytoplasm of mammalian cells. In mammalian cell nuclei, however, the impact of RNAi has remained more controversial. A key technical hurdle has been a lack of optimized protocols for the isolation and analysis of cell nuclei. Here we describe a simplified protocol for nuclei isolation from cultured cells that incorporates a method for obtaining nucleoplasmic and chromatin fractions and removing cytoplasmic contamination. Cell fractions can then be used to detect the presence and activity of RNAi factors in the nucleus. We include a method for investigating an early step in RNAi, Argonaute protein loading with small RNAs, which is enabled by our improved extract preparations. This protocol facilitates the characterization of nuclear RNAi, and it can be applied to the analysis of other nuclear proteins and pathways. From cellular fractionation to analysis of Argonaute loading results, this protocol takes 4–6 d to complete.

Digital Quantitation of Potential Therapeutic Target RNAs

Accurate determination of the amount of a given RNA within a cell is necessary to gain a full understanding of the RNAs function and regulation. Typically, the abundance of RNA is measured by quantitative polymerase chain reaction (qPCR). With qPCR, however, absolute quantification is not possible unless an adequate reference standard curve is generated. The method is not well suited for detecting low copy number templates and values vary depending on the specific primers used. To overcome these drawbacks, digital PCR (dPCR) has been developed to obtain exact values for RNA copies in a sample. Here we report the characterization of droplet digital PCR (ddPCR). We used ddPCR to quantify long noncoding RNAs from various subcellular compartments within human cells and found that results obtained using ddPCR parallel those from qPCR. Mutant huntingtin (HTT) protein is the cause of Huntingtons Disease, and we show that we can quantify human HTT messenger RNA and discriminate between the mutant and wild-type HTT alleles using ddPCR. These results reveal insights into the design of experiments using ddPCR and show that ddPCR can be a robust tool for identifying the number of RNA species inside of cells.

Antisense and antigene inhibition of gene expression by cell-permeable oligonucleotide-oligospermine conjugates.

Oligonucleotides and their derivatives are a proven chemical strategy for modulating gene expression. However, their negative charge remains a challenge for delivery and target recognition inside cells. Here we show that oligonucleotide-oligospermine conjugates (Zip nucleic acids or ZNAs) can help overcome these shortcomings by serving as effective antisense and antigene agents. Conjugates containing DNA and locked nucleic acid (LNA) oligonucleotides are active, and oligospermine conjugation facilitates carrier-free cell uptake at nanomolar concentrations. Conjugates targeting the CAG triplet repeat within huntingtin (HTT) mRNA selectively inhibit expression of the mutant huntingtin protein. Conjugates targeting the promoter of the progesterone receptor (PR) function as antigene agents to block PR expression. These observations support further investigation of ZNA conjugates as gene silencing agents.

Electrophoretic Mobility Shift Assay for Characterizing RNA–Protein Interaction

Electrophoretic mobility shift assay, or EMSA, is a well-established technique for separating macromolecules under native conditions based on a combination of shape, size, and charge. The use of EMSA can provide both general and specific information concerning the interaction between two macromolecules such as RNA and protein. Here we present a protocol for the practical use of EMSA to assess protein-RNA interactions and ribonucleoprotein (RNP) assembly. The conceptual framework of the assay is discussed along with a step-by-step procedure for the binding of archaeal ribosomal protein L7Ae to a box C/D sRNA. Potential pitfalls and common mistakes to avoid are emphasized with technical tips and a notes section. This protocol provides a starting point for the design and implementation of EMSA in studying a wide variety of RNP complexes.