Artificial miRNAs targeting CAG repeat expansion in ORFs cause rapid deadenylation and translation inhibition of mutant transcripts.

Polyglutamine (polyQ) diseases are incurable neurological disorders caused by CAG repeat expansion in the open reading frames (ORFs) of specific genes. This type of mutation in the HTT gene is responsible for Huntington's disease (HD). CAG repeat-targeting artificial miRNAs (art-miRNAs) were shown as attractive therapeutic approach for polyQ disorders as they caused allele-selective decrease in the level of mutant proteins. Here, using polyQ disease models, we aimed to demonstrate how miRNA-based gene expression regulation is dependent on target sequence features. We show that the silencing efficiency and selectivity of art-miRNAs is influenced by the localization of the CAG repeat tract within transcript and the specific sequence context. Furthermore, we aimed to reveal the events leading to downregulation of mutant polyQ proteins and found very rapid activation of translational repression and HTT transcript deadenylation. Slicer-activity of AGO2 was dispensable in this process, as determined in AGO2 knockout cells generated with CRISPR-Cas9 technology. We also showed highly allele-selective downregulation of huntingtin in human HD neural progenitors (NPs). Taken together, art-miRNA activity may serve as a model of the cooperative activity and targeting of ORF regions by endogenous miRNAs.

qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites.

Genome editing technology based on engineered nucleases has been increasingly applied for targeted modification of genes in a variety of cell types and organisms. However, the methods currently used for evaluating the editing efficiency still suffer from many limitations, including preferential detection of some mutation types, sensitivity to polymorphisms that hamper mismatch detection, lack of multiplex capability, or sensitivity to assay conditions. Here, we describe qEva-CRISPR, a new quantitative method that overcomes these limitations and allows simultaneous (multiplex) analysis of CRISPR/Cas9-induced modifications in a target and the corresponding off-targets or in several different targets. We demonstrate all of the advantages of the qEva-CRISPR method using a number of sgRNAs targeting the TP53, VEGFA, CCR5, EMX1 and HTT genes in different cell lines and under different experimental conditions. Unlike other methods, qEva-CRISPR detects all types of mutations, including point mutations and large deletions, and its sensitivity does not depend on the mutation type. Moreover, this approach allows for successful analysis of targets located in 'difficult' genomic regions. In conclusion, qEva-CRISPR may become a method of choice for unbiased sgRNA screening to evaluate experimental conditions that affect genome editing or to distinguish homology-directed repair from non-homologous end joining.

Nuclear speckles: molecular organization, biological function and role in disease.

The nucleoplasm is not homogenous; it consists of many types of nuclear bodies, also known as nuclear domains or nuclear subcompartments. These self-organizing structures gather machinery involved in various nuclear activities. Nuclear speckles (NSs) or splicing speckles, also called interchromatin granule clusters, were discovered as sites for splicing factor storage and modification. Further studies on transcription and mRNA maturation and export revealed a more general role for splicing speckles in RNA metabolism. Here, we discuss the functional implications of the localization of numerous proteins crucial for epigenetic regulation, chromatin organization, DNA repair and RNA modification to nuclear speckles. We highlight recent advances suggesting that NSs facilitate integrated regulation of gene expression. In addition, we consider the influence of abundant regulatory and signaling proteins, i.e. protein kinases and proteins involved in protein ubiquitination, phosphoinositide signaling and nucleoskeletal organization, on pre-mRNA synthesis and maturation. While many of these regulatory proteins act within NSs, direct evidence for mRNA metabolism events occurring in NSs is still lacking. NSs contribute to numerous human diseases, including cancers and viral infections. In addition, recent data have demonstrated close relationships between these structures and the development of neurological disorders.

A potential role of extended simple sequence repeats in competing endogenous RNA crosstalk.

MicroRNA (miRNA)-mediated crosstalk between coding and non-coding RNAs of various types is known as the competing endogenous RNA (ceRNA) concept. Here, we propose that there is a specific variant of the ceRNA language that takes advantage of simple sequence repeat (SSR) wording. We applied bioinformatics tools to identify human transcripts that may be regarded as repeat-associated ceRNAs (raceRNAs). Multiple protein-coding transcripts, transcribed pseudogenes, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) showing this potential were identified, and numerous miRNAs were predicted to bind to SSRs. We propose that simple repeats expanded in various hereditary neurological diseases may act as sponges for miRNAs containing complementary repeats that would affect raceRNA crosstalk. Based on the representation of specific SSRs in transcripts, expression data for SSR-binding miRNAs and expression profiling data from patients, we determined that raceRNA crosstalk is most likely to be perturbed in the case of myotonic dystrophy type 1 (DM1) and type 2 (DM2).

2D and 3D FISH of expanded repeat RNAs in human lymphoblasts.

The first methods for visualizing RNAs within cells were designed for simple imaging of specific transcripts in cells or tissues and since then significant technical advances have been made in this field. Today, high-resolution images can be obtained, enabling visualization of single transcript molecules, quantitative analyses of images, and precise localization of RNAs within cells as well as co-localization of transcripts with specific proteins or other molecules. In addition, tracking of RNA dynamics within single cell has become possible. RNA imaging techniques have been utilized for investigating the role of mutant RNAs in a number of human disorders caused by simple microsatellite expansions. These diseases include myotonic dystrophy type 1 and 2, amyotrophic lateral sclerosis/frontotemporal dementia, fragile X-associated tremor/ataxia syndrome, and Huntington's disease. Mutant RNAs with expanded repeats tend to aggregate predominantly within cell nuclei, forming structures called RNA foci. In this study, we demonstrate methods for fluorescent visualization of RNAs in both fixed and living cells using the example of RNAs containing various expanded repeat tracts (CUG, CCUG, GGGGCC, CGG, and CAG) from experiment design to image analysis. We describe in detail 2D and 3D fluorescence in situ hybridization (FISH) protocols for imaging expanded repeats RNAs, and we review briefly live imaging techniques used to characterize RNA foci formed by mutant RNAs. These methods could be used to image the entire cellular pathway of RNAs, from transcription to degradation.

Identifying proteins that bind to specific RNAs - focus on simple repeat expansion diseases.

RNA-protein complexes play a central role in the regulation of fundamental cellular processes, such as mRNA splicing, localization, translation and degradation. The misregulation of these interactions can cause a variety of human diseases, including cancer and neurodegenerative disorders. Recently, many strategies have been developed to comprehensively analyze these complex and highly dynamic RNA-protein networks. Extensive efforts have been made to purify in vivo-assembled RNA-protein complexes. In this review, we focused on commonly used RNA-centric approaches that involve mass spectrometry, which are powerful tools for identifying proteins bound to a given RNA. We present various RNA capture strategies that primarily depend on whether the RNA of interest is modified. Moreover, we briefly discuss the advantages and limitations of in vitro and in vivo approaches. Furthermore, we describe recent advances in quantitative proteomics as well as the methods that are most commonly used to validate robust mass spectrometry data. Finally, we present approaches that have successfully identified expanded repeat-binding proteins, which present abnormal RNA-protein interactions that result in the development of many neurological diseases.

miRNAmotif-A Tool for the Prediction of Pre-miRNA⁻Protein Interactions.

MicroRNAs (miRNAs) are short, non-coding post-transcriptional gene regulators. In mammalian cells, mature miRNAs are produced from primary precursors (pri-miRNAs) using canonical protein machinery, which includes Drosha/DGCR8 and Dicer, or the non-canonical mirtron pathway. In plant cells, mature miRNAs are excised from pri-miRNAs by the DICER-LIKE1 (DCL1) protein complex. The involvement of multiple regulatory proteins that bind directly to distinct miRNA precursors in a sequence- or structure-dependent manner adds to the complexity of the miRNA maturation process. Here, we present a web server that enables searches for miRNA precursors that can be recognized by diverse RNA-binding proteins based on known sequence motifs to facilitate the identification of other proteins involved in miRNA biogenesis. The database used by the web server contains known human, murine, and Arabidopsis thaliana pre-miRNAs. The web server can also be used to predict new RNA-binding protein motifs based on a list of user-provided sequences. We show examples of miRNAmotif applications, presenting precursors that contain motifs recognized by Lin28, MCPIP1, and DGCR8 and predicting motifs within pre-miRNA precursors that are recognized by two DEAD-box helicases-DDX1 and DDX17. miRNAmotif is released as an open-source software under the MIT License. The code is available at GitHub (www.github.com/martynaut/mirnamotif). The webserver is freely available at http://mirnamotif.ibch.poznan.pl.

Nuclear speckles are detention centers for transcripts containing expanded CAG repeats.

The human genetic disorders caused by CAG repeat expansions in the translated sequences of various genes are called polyglutamine (polyQ) diseases because of the cellular "toxicity" of the mutant proteins. The contribution of mutant transcripts to the pathogenesis of these diseases is supported by several observations obtained from cellular models of these disorders. Here, we show that the common feature of cell lines modeling polyQ diseases is the formation of nuclear CAG RNA foci. We performed qualitative and quantitative analyses of these foci in numerous cellular models endogenously and exogenously expressing mutant transcripts by fluorescence in situ hybridization (FISH). We compared the CAG RNA foci of polyQ diseases with the CUG foci of myotonic dystrophy type 1 and found substantial differences in their number and morphology. Smaller differences within the polyQ disease group were also revealed and included a positive correlation between the foci number and the CAG repeat length. We show that expanded CAA repeats, also encoding glutamine, did not trigger RNA foci formation and foci formation is independent of the presence of mutant polyglutamine protein. Using FISH combined with immunofluorescence, we demonstrated partial co-localization of CAG repeat foci with MBNL1 alternative splicing factor, which explains the mild deregulation of MBNL1-dependent genes. We also showed that foci reside within nuclear speckles in diverse cell types: fibroblasts, lymphoblasts, iPS cells and neuronal progenitors and remain dependent on integrity of these nuclear structures.

Sequence-non-specific effects generated by various types of RNA interference triggers.

RNA interference triggers such as short interfering RNA (siRNA) or genetically encoded short hairpin RNA (shRNA) and artificial miRNA (sh-miR) are widely used to silence the expression of specific genes. In addition to silencing selected targets, RNAi reagents may induce various side effects, including immune responses. To determine the molecular markers of immune response activation when using RNAi reagents, we analyzed the results of experiments gathered in the RNAimmuno (v 2.0) and GEO Profiles databases. To better characterize and compare cellular responses to various RNAi reagents in one experimental system, we designed a reagent series in corresponding siRNA, D-siRNA, shRNA and sh-miR forms. To exclude sequence-specific effects the reagents targeted 3 different transcripts (Luc, ATXN3 and HTT). We demonstrate that RNAi reagents induce a broad variety of sequence-non-specific effects, including the deregulation of cellular miRNA levels. Typical siRNAs are weak stimulators of interferon response but may saturate the miRNA biogenesis pathway, leading to the downregulation of highly expressed miRNAs, whereas plasmid-based reagents induce known markers of immune response and may alter miRNA levels and their isomiR composition.

siRNA release from pri-miRNA scaffolds is controlled by the sequence and structure of RNA.

shmiRs are pri-miRNA-based RNA interference triggers from which exogenous siRNAs are expressed in cells to silence target genes. These reagents are very promising tools in RNAi in vivo applications due to their good activity profile and lower toxicity than observed for other vector-based reagents such as shRNAs. In this study, using high-resolution northern blotting and small RNA sequencing, we investigated the precision with which RNases Drosha and Dicer process shmiRs. The fidelity of siRNA release from the commonly used pri-miRNA shuttles was found to depend on both the siRNA insert and the pri-miR scaffold. Then, we searched for specific factors that may affect the precision of siRNA release and found that both the structural features of shmiR hairpins and the nucleotide sequence at Drosha and Dicer processing sites contribute to cleavage site selection and cleavage precision. An analysis of multiple shRNA intermediates generated from several reagents revealed the complexity of shmiR processing by Drosha and demonstrated that Dicer selects substrates for further processing. Aside from providing new basic knowledge regarding the specificity of nucleases involved in miRNA biogenesis, our results facilitate the rational design of more efficient genetic reagents for RNAi technology.

RNA FISH for detecting expanded repeats in human diseases.

RNA fluorescence in situ hybridization (FISH) is a widely used technique for detecting transcripts in fixed cells and tissues. Many variants of RNA FISH have been proposed to increase signal strength, resolution and target specificity. The current variants of this technique facilitate the detection of the subcellular localization of transcripts at a single molecule level. Among the applications of RNA FISH are studies on nuclear RNA foci in diseases resulting from the expansion of tri-, tetra-, penta- and hexanucleotide repeats present in different single genes. The partial or complete retention of mutant transcripts forming RNA aggregates within the nucleoplasm has been shown in multiple cellular disease models and in the tissues of patients affected with these atypical mutations. Relevant diseases include, among others, myotonic dystrophy type 1 (DM1) with CUG repeats, Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3) with CAG repeats, fragile X-associated tremor/ataxia syndrome (FXTAS) with CGG repeats, myotonic dystrophy type 2 (DM2) with CCUG repeats, amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) with GGGGCC repeats and spinocerebellar ataxia type 32 (SCA32) with GGCCUG. In this article, we summarize the results obtained with FISH to examine RNA nuclear inclusions. We provide a detailed protocol for detecting RNAs containing expanded CAG and CUG repeats in different cellular models, including fibroblasts, lymphoblasts, induced pluripotent stem cells and murine and human neuronal progenitors. We also present the results of the first single-molecule FISH application in a cellular model of polyglutamine disease.

Modeling simple repeat expansion diseases with iPSC technology.

A number of human genetic disorders, including Huntington's disease, myotonic dystrophy type 1, C9ORF72 form of amyotrophic lateral sclerosis and several spinocerebellar ataxias, are caused by the expansion of various microsatellite sequences in single implicated genes. The neurodegenerative and neuromuscular nature of the repeat expansion disorders considerably limits the access of researchers to appropriate cellular models of these diseases. This limitation, however, can be overcome by the application of induced pluripotent stem cell (iPSC) technology. In this paper, we review the current knowledge on the modeling of repeat expansion diseases with human iPSCs and iPSC-derived cells, focusing on the disease phenotypes recapitulated in these models. In subsequent sections, we provide basic practical knowledge regarding iPSC generation, characterization and differentiation into neurons. We also cover disease modeling in iPSCs, neuronal stem cells and specialized neuronal cultures. Furthermore, we also summarize the therapeutic potential of iPSC technology in repeat expansion diseases.

Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer.

The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3' arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5' end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3' overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3' overhangs.

Cooperation meets competition in microRNA-mediated DMPK transcript regulation.

The fundamental role of microRNAs (miRNAs) in the regulation of gene expression has been well-established, but many miRNA-driven regulatory mechanisms remain elusive. In the present study, we demonstrate that miRNAs regulate the expression of DMPK, the gene mutated in myotonic dystrophy type 1 (DM1), and we provide insight regarding the concerted effect of the miRNAs on the DMPK target. Specifically, we examined the binding of several miRNAs to the DMPK 3' UTR using luciferase assays. We validated the interactions between the DMPK transcript and the conserved miR-206 and miR-148a. We suggest a possible cooperativity between these two miRNAs and discuss gene targeting by miRNA pairs that vary in distance between their binding sites and expression profiles. In the same luciferase reporter system, we showed miR-15b/16 binding to the non-conserved CUG repeat tract present in the DMPK transcript and that the CUG-repeat-binding miRNAs might also act cooperatively. Moreover, we detected miR-16 in cytoplasmic foci formed by exogenously expressed RNAs with expanded CUG repeats. Therefore, we propose that the expanded CUGs may serve as a target for concerted regulation by miRNAs and may also act as molecular sponges for natural miRNAs with CAG repeats in their seed regions, thereby affecting their physiological functions.

Oligonucleotide-based strategies to combat polyglutamine diseases.

Considerable advances have been recently made in understanding the molecular aspects of pathogenesis and in developing therapeutic approaches for polyglutamine (polyQ) diseases. Studies on pathogenic mechanisms have extended our knowledge of mutant protein toxicity, confirmed the toxicity of mutant transcript and identified other toxic RNA and protein entities. One very promising therapeutic strategy is targeting the causative gene expression with oligonucleotide (ON) based tools. This straightforward approach aimed at halting the early steps in the cascade of pathogenic events has been widely tested for Huntington's disease and spinocerebellar ataxia type 3. In this review, we gather information on the use of antisense oligonucleotides and RNA interference triggers for the experimental treatment of polyQ diseases in cellular and animal models. We present studies testing non-allele-selective and allele-selective gene silencing strategies. The latter include targeting SNP variants associated with mutations or targeting the pathologically expanded CAG repeat directly. We compare gene silencing effectors of various types in a number of aspects, including their design, efficiency in cell culture experiments and pre-clinical testing. We discuss advantages, current limitations and perspectives of various ON-based strategies used to treat polyQ diseases.

RAN translation and frameshifting as translational challenges at simple repeats of human neurodegenerative disorders.

Repeat-associated disorders caused by expansions of short sequences have been classified as coding and noncoding and are thought to be caused by protein gain-of-function and RNA gain-of-function mechanisms, respectively. The boundary between such classifications has recently been blurred by the discovery of repeat-associated non-AUG (RAN) translation reported in spinocerebellar ataxia type 8, myotonic dystrophy type 1, fragile X tremor/ataxia syndrome and C9ORF72 amyotrophic lateral sclerosis and frontotemporal dementia. This noncanonical translation requires no AUG start codon and can initiate in multiple frames of CAG, CGG and GGGGCC repeats of the sense and antisense strands of disease-relevant transcripts. RNA structures formed by the repeats have been suggested as possible triggers; however, the precise mechanism of the translation initiation remains elusive. Templates containing expansions of microsatellites have also been shown to challenge translation elongation, as frameshifting has been recognized across CAG repeats in spinocerebellar ataxia type 3 and Huntington's disease. Determining the critical requirements for RAN translation and frameshifting is essential to decipher the mechanisms that govern these processes. The contribution of unusual translation products to pathogenesis needs to be better understood. In this review, we present current knowledge regarding RAN translation and frameshifting and discuss the proposed mechanisms of translational challenges imposed by simple repeat expansions.

A new humanized ataxin-3 knock-in mouse model combines the genetic features, pathogenesis of neurons and glia and late disease onset of SCA3/MJD.

Spinocerebellar ataxia type 3 (SCA3/MJD) is a neurodegenerative disease triggered by the expansion of CAG repeats in the ATXN3 gene. Here, we report the generation of the first humanized ataxin-3 knock-in mouse model (Ki91), which provides insights into the neuronal and glial pathology of SCA3/MJD. First, mutant ataxin-3 accumulated in cell nuclei across the Ki91 brain, showing diffused immunostaining and forming intranuclear inclusions. The humanized allele revealed expansion and contraction of CAG repeats in intergenerational transmissions. CAG mutation also exhibited age-dependent tissue-specific expansion, which was most prominent in the cerebellum, pons and testes of Ki91 animals. Moreover, Ki91 mice displayed neuroinflammatory processes, showing astrogliosis in the cerebellar white matter and the substantia nigra that paralleled the transcriptional deregulation of Serpina3n, a molecular sign of neurodegeneration and brain damage. Simultaneously, the cerebellar Purkinje cells in Ki91 mice showed neurodegeneration, a pronounced decrease in Calbindin D-28k immunoreactivity and a mild decrease in cell number, thereby modeling the degeneration of the cerebellum observed in SCA3. Moreover, these molecular and cellular neuropathologies were accompanied by late behavioral deficits in motor coordination observed in rotarod and static rod tests in heterozygous Ki91 animals. In summary, we created an ataxin-3 knock-in mouse model that combines the molecular and behavioral disease phenotypes with the genetic features of SCA3. This model will be very useful for studying the pathogenesis and responses to therapy of SCA3/MJD and other polyQ disorders.

Characterization of a naturally occurring truncated Dicer.

Dicer is central to small RNA silencing pathways, thus playing an important role in physiological and pathological states. Recently, a number of mutations in dicer gene have been identified in diverse types of cancer, implicating Dicer in oncogenic cooperation. Here we report on the properties of a rare splice variant of the human dicer gene, occurring in neuroblastoma cells, and not detectable in normal tissues. Due to the skipping of one exon, the alternatively spliced transcript encodes a putative truncated protein, t-Dicer, lacking the dsRNA-binding domain and bearing altered one of the two RNase III catalytic centers. The ability of the exon-depleted t-dicer transcript to be translated in vitro was first investigated by the expression of flagged t-Dicer in human cells. We found that t-dicer transcript could be translated in vitro, albeit not as efficiently as full-length dicer transcript. Then, the possible enzymatic activity of t-Dicer was analyzed by an in vitro dicing assay able to distinguish the enzymatic activity of the individual RNase III domains. We showed that t-Dicer preserved partial dicing activity. Overall, the results indicate that t-dicer transcript could produce a protein still able to bind the substrate and to cleave only one of the two pre-miRNA strands. Given the increasing number of mutations reported for dicer gene in tumours, our experimental approach could be useful to characterize the activity of these mutants, which may dictate changes in selected classes of small RNAs and/or lead to their aberrant maturation.

The panorama of miRNA-mediated mechanisms in mammalian cells.

MicroRNAs comprise a large family of short, non-coding RNAs that are present in most eukaryotic organisms and are typically involved in downregulating the expression of protein-coding genes. The detailed mechanisms of miRNA functioning in animals and plants have been under investigation for more than decade. In mammalian cells, miRNA guides the effector complex miRISC to bind with partially complementary sequences, usually within the 3'UTR of mRNAs, and inhibit protein synthesis with or without transcript degradation. In addition to these main mechanisms, several other modes of miRNA-mediated gene expression regulation have been described, but their scale and importance remain a matter of debate. In this review, we briefly summarize the pathway of miRNA precursor processing during miRNA biogenesis and continue with the description of the miRISC assembly process. Then, we present the miRNA-mediated mechanisms of gene expression regulation in detail, and we gather information concerning the proteins involved in these processes. In addition, we briefly refer to the current applications of miRNA mechanisms in therapeutic strategies. Finally, we highlight some of the remaining controversies surrounding the regulation of mammalian gene expression by miRNAs.

Self-duplexing CUG repeats selectively inhibit mutant huntingtin expression.

Huntington's disease (HD) is a neurodegenerative genetic disorder caused by the expansion of the CAG repeat in the translated sequence of the HTT gene. This expansion generates a mutant huntingtin protein that contains an abnormally elongated polyglutamine tract, which, together with mutant transcript, causes cellular dysfunction. Currently, there is no curative treatment available to patients suffering from HD; however, the selective inhibition of the mutant allele expression is a promising therapeutic option. In this study, we developed a new class of CAG repeat-targeting silencing reagents that consist of self-duplexing CUG repeats. Self-duplex formation was induced through one or several U-base substitutions. A number of self-duplexing guide-strand-only short interfering RNAs have been tested through transfection into cells derived from HD patients, showing distinct activity profiles. The best reagents were highly discriminatory between the normal and mutant HTT alleles (allele selectivity) and the HTT transcript and other transcripts containing shorter CAG repeats (gene selectivity). We also demonstrated that the self-duplexing CUG repeat short interfering RNAs use the RNA interference pathway to elicit silencing, and repeat-targeting reagents showed similar activity and selectivity when expressed from short hairpin RNA vectors to achieve more durable silencing effects.