Marzena Wojciechowska

Cellular toxicity of expanded RNA repeats: focus on RNA foci

Discrete and punctate nuclear RNA foci are characteristic molecular hallmarks of pathogenesis in myotonic dystrophy type 1 and type 2. Intranuclear RNA inclusions of distinct morphology have also been found in fragile X-associated tremor ataxia syndrome, Huntingtons disease-like 2, spinocerebellar ataxias type 8, type 10 and type 31. These neurological diseases are associated with the presence of abnormally long simple repeat expansions in their respective genes whose expression leads to the formation of flawed transcripts with altered metabolisms. Expanded CUG, CCUG, CGG, CAG, AUUCU and UGGAA repeats are associated with the diseases and accumulate in nuclear foci, as demonstrated in variety of cells and tissues of human and model organisms. These repeat RNA foci differ in size, shape, cellular abundance and protein composition and their formation has a negative impact on cellular functions. This review summarizes the efforts of many laboratories over the past 15 years to characterize nuclear RNA foci that are recognized as important triggers in the mutant repeat RNA toxic gain-of-function mechanisms of pathogenesis in neurological disorders.

Mutant CAG repeats of Huntingtin transcript fold into hairpins, form nuclear foci and are targets for RNA interference

The CAG repeat expansions that occur in translated regions of specific genes can cause human genetic disorders known as polyglutamine (poly-Q)-triggered diseases. Huntington’s disease and spinobulbar muscular atrophy (SBMA) are examples of these diseases in which underlying mutations are localized near other trinucleotide repeats in the huntingtin (HTT) and androgen receptor (AR) genes, respectively. Mutant proteins that contain expanded polyglutamine tracts are well-known triggers of pathogenesis in poly-Q diseases, but a toxic role for mutant transcripts has also been proposed. To gain insight into the structural features of complex triplet repeats of HTT and AR transcripts, we determined their structures in vitro and showed the contribution of neighboring repeats to CAG repeat hairpin formation. We also demonstrated that the expanded transcript is retained in the nucleus of human HD fibroblasts and is colocalized with the MBNL1 protein. This suggests that the CAG repeats in the HTT mRNA adopt ds-like RNA conformations in vivo. The intracellular structure of the CAG repeat region of mutant HTT transcripts was not sufficiently stable to be protected from cleavage by an siRNA targeting the repeats and the silencing efficiency was higher for the mutant transcript than for its normal counterpart.

CAG repeats mimic CUG repeats in the misregulation of alternative splicing

Mutant transcripts containing expanded CUG repeats in the untranslated region are a pathogenic factor in myotonic dystrophy type 1 (DM1). The mutant RNA sequesters the muscleblind-like 1 (MBNL1) splicing factor and causes misregulation of the alternative splicing of multiple genes that are linked to clinical symptoms of the disease. In this study, we show that either long untranslated CAG repeat RNA or short synthetic CAG repeats induce splicing aberrations typical of DM1. Alternative splicing defects are also caused by translated CAG repeats in normal cells transfected with a mutant ATXN3 gene construct and in cells derived from spinocerebellar ataxia type 3 and Huntingtons disease patients. Splicing misregulation is unlikely to be caused by traces of antisense transcripts with CUG repeats, and the possible trigger of this misregulation may be sequestration of the MBNL1 protein with nuclear RNA inclusions containing expanded CAG repeat transcripts. We propose that alternative splicing misregulation by mutant CAG repeats may contribute to the pathological features of polyglutamine disorders.

Triplet repeat RNA structure and its role as pathogenic agent and therapeutic target

This review presents detailed information about the structure of triplet repeat RNA and addresses the simple sequence repeats of normal and expanded lengths in the context of the physiological and pathogenic roles played in human cells. First, we discuss the occurrence and frequency of various trinucleotide repeats in transcripts and classify them according to the propensity to form RNA structures of different architectures and stabilities. We show that repeats capable of forming hairpin structures are overrepresented in exons, which implies that they may have important functions. We further describe long triplet repeat RNA as a pathogenic agent by presenting human neurological diseases caused by triplet repeat expansions in which mutant RNA gains a toxic function. Prominent examples of these diseases include myotonic dystrophy type 1 and fragile X-associated tremor ataxia syndrome, which are triggered by mutant CUG and CGG repeats, respectively. In addition, we discuss RNA-mediated pathogenesis in polyglutamine disorders such as Huntingtons disease and spinocerebellar ataxia type 3, in which expanded CAG repeats may act as an auxiliary toxic agent. Finally, triplet repeat RNA is presented as a therapeutic target. We describe various concepts and approaches aimed at the selective inhibition of mutant transcript activity in experimental therapies developed for repeat-associated diseases.

CAG repeat RNA as an auxiliary toxic agent in polyglutamine disorders.

Over 20 genetic loci with abnormal expansions of short tandem repeats have been associated with human hereditary neurological diseases. Of these, specific trinucleotide repeats located in non-coding and coding regions of individual genes implicated in these disorders are strongly overrepresented. Expansions of CTG, CGG and CAG repeats are linked to, respectively, myotonic dystrophy type 1 (DM1), fragile X-associated tremor/ataxia syndrome (FXTAS), as well as Huntington’s disease (HD) and a number of spinocerebellar ataxias (SCAs). Expanded CAG repeats in translated exons trigger the most disorders for which a protein gain-of-function mechanism has been proposed to explain neurodegeneration by polyglutamine-rich (poly-Q) proteins. However, the results of last years showed that RNA composed of mutated CAG repeats can also be toxic and contribute to pathogenesis of polyglutamine disorders through an RNA-mediated gain-of-function mechanism. This mechanism has been best characterized in the non-coding repeat disorder DM1 and is also implicated in several other diseases, such as FXTAS, spinocerebellar ataxia type 8 (SCA8), Huntingtons disease-like 2 (HDL2), as well as in myotonic dystrophy type 2 (DM2), spinocerebellar ataxia type 10 (SCA10) and type 31 (SCA31). In this review, we summarize recent findings that emphasize the participation of coding mutant CAG repeat RNA in the pathogenesis of polyglutamine disorders, and we discuss the basis of an RNA gain-of-function model in non-coding diseases such as DM1, FXTAS and SCA8.

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 Huntingtons 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.

Small molecule kinase inhibitors alleviate different molecular features of myotonic dystrophy type 1

Expandable (CTG)n repeats in the 3′ UTR of the DMPK gene are a cause of myotonic dystrophy type 1 (DM1), which leads to a toxic RNA gain-of-function disease. Mutant RNAs with expanded CUG repeats are retained in the nucleus and aggregate in discrete inclusions. These foci sequester splicing factors of the MBNL family and trigger upregulation of the CUGBP family of proteins resulting in the mis-splicing of their target transcripts. To date, many efforts to develop novel therapeutic strategies have been focused on disrupting the toxic nuclear foci and correcting aberrant alternative splicing via targeting mutant CUG repeats RNA; however, no effective treatment for DM1 is currently available. Herein, we present results of culturing of human DM1 myoblasts and fibroblasts with two small-molecule ATP-binding site-specific kinase inhibitors, C16 and C51, which resulted in the alleviation of the dominant-negative effects of CUG repeat expansion. Reversal of the DM1 molecular phenotype includes a reduction of the size and number of foci containing expanded CUG repeat transcripts, decreased steady-state levels of CUGBP1 protein, and consequent improvement of the aberrant alternative splicing of several pre-mRNAs misregulated in DM1.

Oncogenomic portals for the visualization and analysis of genome-wide cancer data

Somatically acquired genomic alterations that drive oncogenic cellular processes are of great scientific and clinical interest. Since the initiation of large-scale cancer genomic projects (e.g., the Cancer Genome Project, The Cancer Genome Atlas, and the International Cancer Genome Consortium cancer genome projects), a number of web-based portals have been created to facilitate access to multidimensional oncogenomic data and assist with the interpretation of the data. The portals provide the visualization of small-size mutations, copy number variations, methylation, and gene/protein expression data that can be correlated with the available clinical, epidemiological, and molecular features. Additionally, the portals enable to analyze the gathered data with the use of various user-friendly statistical tools. Herein, we present a highly illustrated review of seven portals, i.e., Tumorscape, UCSC Cancer Genomics Browser, ICGC Data Portal, COSMIC, cBioPortal, IntOGen, and BioProfiling.de. All of the selected portals are user-friendly and can be exploited by scientists from different cancer-associated fields, including those without bioinformatics background. It is expected that the use of the portals will contribute to a better understanding of cancer molecular etiology and will ultimately accelerate the translation of genomic knowledge into clinical practice.

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.

The 30 kb deletion in the APOBEC3 cluster decreases APOBEC3A and APOBEC3B expression and creates a transcriptionally active hybrid gene but does not associate with breast cancer in the European population

APOBEC3B, in addition to other members of the APOBEC3 gene family, has recently been intensively studied due to its identification as a gene whose activation in cancer is responsible for a specific pattern of massively occurring somatic mutations. It was recently shown that a common large deletion in the APOBEC3 cluster (the APOBEC3B deletion) may increase the risk of breast cancer. However, conflicting evidence regarding this association was also reported. In the first step of our study, using different approaches, including an in-house designed multiplex ligation-dependent probe amplification assay, we analyzed the structure of the deletion and showed that although the breakpoints are located in highly homologous regions, which may generate recurrent occurrence of similar but not identical deletions, there is no sign of deletion heterogeneity. This knowledge allowed us to distinguish transcripts of all affected genes, including the highly homologous canonical APOBEC3A and APOBEC3B, and the hybrid APOBEC3A/APOBEC3B gene. We unambiguously confirmed the presence of the hybrid transcript and showed that the APOBEC3B deletion negatively correlates with APOBEC3A and APOBEC3B expression and positively correlates with APOBEC3A/APOBEC3B expression, whose mRNA level is >10-fold and >1500-fold lower than the level of APOBEC3A and APOBEC3B, respectively. In the next step, we performed a large-scale association study in three different cohorts (2972 cases and 3682 controls) and showed no association of the deletion with breast cancer, familial breast cancer or ovarian cancer. Further, we conducted a meta-analysis that confirmed the lack of the association of the deletion with breast cancer in non-Asian populations.APOBEC3B, in addition to other members of the APOBEC3 gene family, has recently been intensively studied due to its identification as a gene whose activation in cancer is responsible for a specific pattern of massively occurring somatic mutations. It was recently shown that a common large deletion in the APOBEC3 cluster (the APOBEC3B deletion) may increase the risk of breast cancer. However, conflicting evidence regarding this association was also reported. In the first step of our study, using different approaches, including an in-house designed multiplex ligation-dependent probe amplification assay, we analyzed the structure of the deletion and showed that although the breakpoints are located in highly homologous regions, which may generate recurrent occurrence of similar but not identical deletions, there is no sign of deletion heterogeneity. This knowledge allowed us to distinguish transcripts of all affected genes, including the highly homologous canonical APOBEC3A and APOBEC3B, and the hybrid APOBEC3A/APOBEC3B gene. We unambiguously confirmed the presence of the hybrid transcript and showed that the APOBEC3B deletion negatively correlates with APOBEC3A and APOBEC3B expression and positively correlates with APOBEC3A/APOBEC3B expression, whose mRNA level is >10-fold and >1500-fold lower than the level of APOBEC3A and APOBEC3B, respectively. In the next step, we performed a large-scale association study in three different cohorts (2972 cases and 3682 controls) and showed no association of the deletion with breast cancer, familial breast cancer or ovarian cancer. Further, we conducted a meta-analysis that confirmed the lack of the association of the deletion with breast cancer in non-Asian populations.