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Friedreich's ataxia (FRDA) is a genetic neurodegenerative disease that is caused by guanine-adenine-adenine (GAA) nucleotide repeat expansions in the first intron of the frataxin (FXN) gene. Although present in the intron, this mutation leads to a substantial decrease in protein expression. Currently, no effective treatment is available for FRDA, and, in addition to FXN, other targets with therapeutic potential are continuously sought. As miRNAs can regulate the expression of a broad spectrum of genes, are used as biomarkers, and can serve as therapeutic tools, we decided to identify and characterize differentially expressed miRNAs and their targets in FRDA cells compared to unaffected control (CTRL) cells. In this study, we performed an integrated miRNAseq and RNAseq analysis using the same cohort of primary FRDA and CTRL cells. The results of the transcriptome studies were supported by bioinformatic analyses and validated by qRT-PCR. miRNA interactions with target genes were assessed by luciferase assays, qRT-PCR, and immunoblotting. In silico analysis identified the FXN transcript as a target of five miRNAs upregulated in FRDA cells. Further studies confirmed that miRNA-224-5p indeed targets FXN, resulting in decreases in mRNA and protein levels. We also validated the ability of miRNA-10a-5p to bind and regulate the levels of brain-derived neurotrophic factor (BDNF), an important modulator of neuronal growth. We observed a significant decrease in the levels of miRNA-10a-5p and increase in the levels of BDNF upon correction of FRDA cells via zinc-finger nuclease (ZFN)-mediated excision of expanded GAA repeats. Our comprehensive transcriptome analyses identified miRNA-224-5p and miRNA-10a-5p as negative regulators of the FXN and BDNF expression, respectively. These results emphasize not only the importance of miRNAs in the pathogenesis of FRDA but also their potential as therapeutic targets for this disease.

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ACKNOWLEDGEMENTSThis work is dedicated to the memory of Professor Wlodzimierz J. Krzyzosiak. We thank Professor Marta Olejniczak from Department of Genome Engineering for reviewing the manuscript and every day stimulating discussions. We thank Professor Jerzy Ciesiolka from Department of RNA Biochemistry for helpful suggestions. The confocal microscopy analyses were performed in the ABSTRACT Spinocerebellar ataxia type 3 (SCA3) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene encoding the ataxin-3 protein. Despite extensive research the exact pathogenic mechanisms of SCA3 are still not understood in depth. In the present study, to gain insight into the toxicity induced by the expanded CAG repeats in SCA3, we comprehensively investigated repeat-associated non-ATG (RAN) translation in various cellular models expressing translated or non-canonically translated ATXN3 sequences with an increasing number of CAG repeats.We demonstrate that two SCA3 RAN proteins, polyglutamine (polyQ) and polyalanine (polyA), are found only in the case of CAG repeats of pathogenic length. Despite having distinct cellular localization, RAN polyQ and RAN polyA proteins are very often coexpressed in the same cell, impairing nuclear integrity and inducing apoptosis. We provide for the first time mechanistic insights into SCA3 RAN translation indicating that ATXN3 sequences surrounding the repeat region have an impact on SCA3 RAN translation initiation and efficiency. We revealed that RAN translation of polyQ proteins starts at noncognate codons upstream of the CAG repeats, whereas RAN polyA proteins are likely translated within repeats. Furthermore, integrated stress response activation enhances SCA3 RAN translation. We suggest that RAN translation in SCA3 is a common event substantially contributing to SCA3 pathogenesis and that the ATXN3 sequence context plays an important role in triggering this unconventional translation. KEYWORDSSCA3, CAG repeat expansion, RAN translation, translation initiation, non-cognate initiation codon, integrated stress response ABBREVIATIONS ATXN3 -ataxin-3, c9ALS/FTD -c9orf72 amyotrophic lateral sclerosis/frontotemporal dementia, CHOP -C/EBP homologous protein, DM1 -myotonic dystrophy type 1 and type 2, ER -endoplasmic reticulum, FECD -Fuchs' endothelial corneal dystrophy, FXTAS -fragile X tremor ataxia syndrome, iMet-tRNAinitiator Met-tRNA, ISR -integrated stress response, MetAPs -methionine aminopeptidases, NAT -Nterminal acetyltransferase, NPC -nuclear pore complex, polyApolyalanine, polyQpolyglutamine, polySpolyserine, RAN translation -repeat associated non-ATG translation, SA -sodium arsenite, SCA3 -spinocerebellar ataxia type 3, TG -thapsigargin INTRODUCTIONSpinocerebellar ataxia type 3 (SCA3) is the most common form of dominantly inherited ataxia in the world and one of nine polyglutamine (polyQ) neurodegenerative diseases. This progressive and fatal disorder is primarily characterized by neuronal dysfunction and degeneration of the cerebellum and functionall...