Martyna O. Urbanek

Triplet repeats in transcripts: structural insights into RNA toxicity.

Abstract Tandem repeats of various trinucleotide motifs are frequent entities in transcripts, and RNA structures formed by these sequences depend on the motif type and number of reiterations. The functions performed by normal triplet repeats in transcripts are poorly understood, but abnormally expanded repeats of certain types trigger pathogenesis in several human genetic disorders known as the triplet repeat expansion diseases (TREDs). The diseases caused by expanded non-coding CUG and CGG repeats in transcripts include myotonic dystrophy type 1 and fragile X-associated tremor ataxia syndrome. Another group of disorders in which transcripts containing translated CAG repeats play an auxiliary role in pathogenesis include Huntington’s disease and several spinocerebellar ataxias. In this review, we gathered existing knowledge regarding the structural features of triplet repeats in transcripts and discussed this in the context of various pathogenic mechanisms assigned to toxic RNA repeats. These mechanisms include aberrant alternative splicing, the inhibition of nuclear transport and export, induction of the innate immune response, alteration of a microRNA biogenesis pathway and abnormal activation of an RNA interference pathway. We also provide ideas for future investigations to reveal further mechanisms of pathogenesis directly triggered by mutant RNA repeats in TREDs.

RNA imaging in living cells – methods and applications

Numerous types of transcripts perform multiple functions in cells, and these functions are mainly facilitated by the interactions of the RNA with various proteins and other RNAs. Insight into the dynamics of RNA biosynthesis, processing and cellular activities is highly desirable because this knowledge will deepen our understanding of cell physiology and help explain the mechanisms of RNA-mediated pathologies. In this review, we discuss the live RNA imaging systems that have been developed to date. We highlight information on the design of these systems, briefly discuss their advantages and limitations and provide examples of their numerous applications in various organisms and cell types. We present a detailed examination of one application of RNA imaging systems: this application aims to explain the role of mutant transcripts in human disease pathogenesis caused by triplet repeat expansions. Thus, this review introduces live RNA imaging systems and provides a glimpse into their various applications.

Small RNA Detection by in Situ Hybridization Methods

Small noncoding RNAs perform multiple regulatory functions in cells, and their exogenous mimics are widely used in research and experimental therapies to interfere with target gene expression. MicroRNAs (miRNAs) are the most thoroughly investigated representatives of the small RNA family, which includes short interfering RNAs (siRNAs), PIWI-associated RNA (piRNAs), and others. Numerous methods have been adopted for the detection and characterization of small RNAs, which is challenging due to their short length and low level of expression. These include molecular biology methods such as real-time RT-PCR, northern blotting, hybridization to microarrays, cloning and sequencing, as well as single cell miRNA detection by microscopy with in situ hybridization (ISH). In this review, we focus on the ISH method, including its fluorescent version (FISH), and we present recent methodological advances that facilitated its successful adaptation for small RNA detection. We discuss relevant technical aspects as well as the advantages and limitations of ISH. We also refer to numerous applications of small RNA ISH in basic research and molecular diagnostics.

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

Abstract 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.

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, Huntingtons 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.

The Role of the Immune System in Triplet Repeat Expansion Diseases

Trinucleotide repeat expansion disorders (TREDs) are a group of dominantly inherited neurological diseases caused by the expansion of unstable repeats in specific regions of the associated genes. Expansion of CAG repeat tracts in translated regions of the respective genes results in polyglutamine- (polyQ-) rich proteins that form intracellular aggregates that affect numerous cellular activities. Recent evidence suggests the involvement of an RNA toxicity component in polyQ expansion disorders, thus increasing the complexity of the pathogenic processes. Neurodegeneration, accompanied by reactive gliosis and astrocytosis is the common feature of most TREDs, which may suggest involvement of inflammation in pathogenesis. Indeed, a number of immune response markers have been observed in the blood and CNS of patients and mouse models, and the activation of these markers was even observed in the premanifest stage of the disease. Although inflammation is not an initiating factor of TREDs, growing evidence indicates that inflammatory responses involving astrocytes, microglia, and the peripheral immune system may contribute to disease progression. Herein, we review the involvement of the immune system in the pathogenesis of triplet repeat expansion diseases, with particular emphasis on polyglutamine disorders. We also present various therapeutic approaches targeting the dysregulated inflammation pathways in these diseases.

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.

Reduction of Huntington’s Disease RNA Foci by CAG Repeat-Targeting Reagents

In several human polyglutamine diseases caused by expansions of CAG repeats in the coding sequence of single genes, mutant transcripts are detained in nuclear RNA foci. In polyglutamine disorders, unlike other repeat-associated diseases, both RNA and proteins exert pathogenic effects; therefore, decreases of both RNA and protein toxicity need to be addressed in proposed treatments. A variety of oligonucleotide-based therapeutic approaches have been developed for polyglutamine diseases, but concomitant assays for RNA foci reduction are lacking. Here, we show that various types of oligonucleotide-based reagents affect RNA foci number in Huntington’s disease cells. We analyzed the effects of reagents targeting either CAG repeat tracts or specific HTT sequences in fibroblasts derived from patients. We tested reagents that either acted as translation blockers or triggered mRNA degradation via the RNA interference pathway or RNase H activation. We also analyzed the effect of chemical modifications of CAG repeat-targeting siRNAs on their efficiency in the foci decline. Our results suggest that the decrease of RNA foci number may be considered as a readout of treatment outcomes for oligonucleotide reagents.

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 Huntingtons 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.