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Stalekar M.,Jozef Stefan Institute | Stalekar M.,King's College London | Yin X.,King's College London | Rebolj K.,Jozef Stefan Institute | And 7 more authors.
Neuroscience | Year: 2015

Transactive response DNA-binding protein 43 (TDP-43) is a predominantly nuclear, ubiquitously expressed RNA and DNA-binding protein. It recognizes and binds to UG repeats and is involved in pre-mRNA splicing, mRNA stability and microRNA metabolism. TDP-43 is essential in early embryonic development but accumulates in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar degeneration (FTLD). It is not known yet whether cytoplasmic aggregates of TDP-43 are toxic or protective but they are often associated with a loss of TDP-43 from the nucleus and neurodegeneration may be caused by a loss of normal TDP-43 function or a gain of toxic function. Here we present a proteomic study to analyze the effect of loss of TDP-43 on the proteome. MS data are available via ProteomeXchange with identifier PXD001668. Our results indicate that TDP-43 is an important regulator of RNA metabolism and intracellular transport. We show that Ran-binding protein 1 (RanBP1), DNA methyltransferase 3 alpha (Dnmt3a) and chromogranin B (CgB) are downregulated upon TDP-43 knockdown. Subsequently, transportin 1 level is increased as a result of RanBP1 depletion. Improper regulation of these proteins and the subsequent disruption of cellular processes may play a role in the pathogenesis of the TDP-43 proteinopathies ALS and FTLD. © 2015 IBRO.


Vatovec S.,Jozef Stefan Institute | Kovanda A.,Jozef Stefan Institute | Kovanda A.,Biomedical Research Institute BRIS | Rogelj B.,Jozef Stefan Institute | Rogelj B.,Biomedical Research Institute BRIS
Neurobiology of Aging | Year: 2014

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are devastating neurodegenerative diseases that form two ends of a complex disease spectrum. Aggregation of RNA binding proteins is one of the hallmark pathologic features of ALS and FTDL and suggests perturbance of the RNA metabolism in their etiology. Recent identification of the disease-associated expansions of the intronic hexanucleotide repeat GGGGCC in the C9ORF72 gene further substantiates the case for RNA involvement. The expanded repeat, which has turned out to be the single most common genetic cause of ALS and FTLD, may enable the formation of complex DNA and RNA structures, changes in RNA transcription, and processing and formation of toxic RNA foci, which may sequester and inactivate RNA binding proteins. Additionally, the transcribed expanded repeat can undergo repeat-associated non-ATG-initiated translation resulting in accumulation of a series of dipeptide repeat proteins. Understanding the basis of the proposed mechanisms and shared pathways, as well as interactions with known key proteins such as TAR DNA-binding protein (TDP-43) are needed to clarify the pathology of ALS and/or FTLD, and make possible steps toward therapy development. © 2014 Elsevier Inc.


Darovic S.,Jozef Stefan Institute | Mihevc S.P.,Jozef Stefan Institute | Zupunski V.,University of Ljubljana | Guncar G.,University of Ljubljana | And 5 more authors.
Journal of Cell Science | Year: 2015

Aberrant cytoplasmic aggregation of FUS, which is caused by mutations primarily in the C-terminal nuclear localisation signal, is associated with 3% of cases of familial amyotrophic lateral sclerosis (ALS). FUS aggregates are also pathognomonic for 10% of all frontotemporal lobar degeneration (FTLD) cases; however, these cases are not associated with mutations in the gene encoding FUS. This suggests that there are differences in the mechanisms that drive inclusion formation of FUS in ALS and FTLD. Here, we show that the C-terminal tyrosine residue at position 526 of FUS is crucial for normal nuclear import. This tyrosine is subjected to phosphorylation, which reduces interaction with transportin 1 and might consequentially affect the transport of FUS into the nucleus. Furthermore, we show that this phosphorylation can occur through the activity of the Src family of kinases. Our study implicates phosphorylation as an additional mechanism by which nuclear transport of FUS might be regulated and potentially perturbed in ALS and FTLD. © 2015. Published by The Company of Biologists Ltd.


Kovanda A.,Jozef Stefan Institute | Kovanda A.,Biomedical Research Institute BRIS | Rezen T.,Biomedical Research Institute BRIS | Rogelj B.,Jozef Stefan Institute | Rogelj B.,Biomedical Research Institute BRIS
Wiley Interdisciplinary Reviews: RNA | Year: 2014

MicroRNAs (miRNAs) are short noncoding RNAs that are important global- as well as tissue- and cell-type-specific regulators of gene expression. Muscle-specific miRNAs or myomirs have been shown to control various processes in skeletal muscles, from myogenesis and muscle homeostasis to different responses to environmental stimuli, such as exercise. Importantly, myomirs are also involved in the development of muscle atrophy arising from aging, immobility, prolonged exposure to microgravity, or muscular and neuromuscular disorders. Additionally, muscle atrophy is both induced by and exacerbates many important chronic and infectious diseases. As global yet specific muscle regulators, myomirs are also good candidates for therapeutic use. Understanding the dynamics of myomirs expression and their role in the development of disease is necessary to determine their potential for muscle atrophy prevention. WIREs RNA 2014, 5:509-525. doi: 10.1002/wrna.1227 For further resources related to this article, please visit the WIREs website. Conflict of interest: The authors have declared no conflicts of interest for this article. © 2014 John Wiley & Sons, Ltd.


Sket P.,Slovenian National Institute of Chemistry | Pohleven J.,Jozef Stefan Institute | Kovanda A.,Jozef Stefan Institute | Kovanda A.,Biomedical Research Institute BRIS | And 7 more authors.
Neurobiology of Aging | Year: 2015

The G4C2 hexanucleotide repeat expansion, located in the first intron of the C9ORF72 gene, represents a major genetic hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several hypotheses have been proposed on how the transcribed repeat RNA leads to the development of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. However, despite their importance, factors affecting the transcription of expanded-repeat RNA are not well known. As transcription is dependent on the DNA containing the expanded repeats, it is crucial to understand its structure. G-quadruplexes are known to affect expression on the level of DNA, therefore whether they form on the expanded-repeat DNA constitutes an important biological question. Using nuclear magnetic resonance and circular dichroism spectroscopy we show that DNA G4C2 with varying number of repeats d(G4C2)n form planar guanine quartets characteristic of G-quadruplexes. Additionally, we show DNA G-quadruplexes can form inter- and intra-molecularly in either parallel or anti-parallel orientation, based on d(G4C2) sequence length. This potential structural heterogeneity of longer disease-relevant repeats should therefore be taken into account when studying their role in disease pathogenesis. © 2015 Elsevier Inc.


Rezen T.,Biomedical Research Institute BRIS | Kovanda A.,Biomedical Research Institute BRIS | Kovanda A.,Jozef Stefan Institute | Eiken O.,KTH Royal Institute of Technology | And 3 more authors.
Acta Physiologica | Year: 2014

Aim: Studies in humans show global changes in mRNA and protein expression occur in human skeletal muscle during bed rest. As microRNAs are important regulators of expression, we analysed the global microRNA expression changes in human muscle following 10 days of sustained bed rest, with the rationale that miRNAs play key roles in atrophy of skeletal muscle. Methods: We analysed expression of miRNA and selected target proteins before and after 10 days of bed rest in biopsies obtained from the vastus lateralis muscle of 6 healthy males. Results: Fifteen of 152 miRNAs detected in human muscle tissue were differentially expressed, and all of them with exception of two were downregulated. The downregulated miRNAs include the following: miR-206, a myomir involved in function and maintenance of skeletal muscle; miR-23a, involved in insulin response and atrophy defence; and several members of the let-7 family involved in cell cycle, cell differentiation and glucose homeostasis. Predicted gene targets of these miRNAs are members of the MAPK, TNF receptor, ALK1, TGF-beta receptor and SMAD signalling pathways. All of these pathways were previously indicated to be involved in skeletal muscle response to physical inactivity. We also measured protein expression of selected miRNA targets and observed a decrease in HDAC4. Conclusion: Our data demonstrate that miRNAs in postural muscles are affected by sustained inactivity and unloading, as induced by prolonged bed rest, and hence are potentially involved in regulation of skeletal muscle adjustments to inactivity. We also propose new miRNAs involved in regulation of biological processes in adult human muscle. © 2014 Scandinavian Physiological Society.

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