title: From transcriptomic to protein level changes in TDP-43 and FUS loss-of-function cell models.
abstract: The full definition of the physiological RNA targets regulated by TDP-43 and FUS RNA-binding proteins (RBPs) represents an important issue in understanding the pathogenic mechanisms associated to these two proteins in amyotrophic lateral sclerosis and frontotemporal dementia. In the last few years several high-throughput screenings have generated a plethora of data, which are difficult to compare due to the different experimental designs and models explored. In this study by using the Affymetrix Exon Arrays, we were able to assess and compare the effects of both TDP-43 and FUS loss-of-function on the whole transcriptome using the same human neuronal SK-N-BE cell model. We showed that TDP-43 and FUS depletion induces splicing and gene expression changes mainly distinct for the two RBPs, although they may regulate common pathways, including neuron differentiation and cytoskeleton organization as evidenced by functional annotation analysis. In particular, TDP-43 and FUS were found to regulate splicing and expression of genes related to neuronal (SEPT6, SULT4A1, TNIK) and RNA metabolism (DICER, ELAVL3/HuC, POLDIP3). Our extended analysis at protein level revealed that these changes have also impact on the protein isoform ratio and content, not always in a direct correlation with transcriptomic data. Contrarily to a loss-of-function mechanism, we showed that mutant TDP-43 proteins maintained their splicing activity in human ALS fibroblasts and experimental cell lines. Our findings further contribute to define the biological functions of these two RBPs in physiological and disease state, strongly encouraging the evaluation of the identified transcriptomic changes at protein level in neuronal experimental models.
authors.affiliation: Department of Pathophysiology and Transplantation, 'Dino Ferrari' Center - Università degli Studi di Milano, Milan 20122, Italy; Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Colombrita
authors.affiliation: Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Onesto
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Trieste 34149, Italy.
authors.forename: Emanuele
authors.initials: E
authors.lastname: Buratti
authors.affiliation: GenoSplice, Hopital Pitié-Salpêtrière, Paris 75013, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: de la Grange
authors.affiliation: Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Gumina
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), AREA Science Park, Trieste 34149, Italy.
authors.forename: Francisco E
authors.initials: FE
authors.lastname: Baralle
authors.affiliation: Department of Pathophysiology and Transplantation, 'Dino Ferrari' Center - Università degli Studi di Milano, Milan 20122, Italy; Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Silani
authors.affiliation: Department of Pathophysiology and Transplantation, 'Dino Ferrari' Center - Università degli Studi di Milano, Milan 20122, Italy; Department of Neurology and Laboratory of Neuroscience, IRCCS Istituto Auxologico Italiano, Milan 20149, Italy. Electronic address: antonia.ratti@unimi.it.
authors.forename: Antonia
authors.initials: A
authors.lastname: Ratti
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D005091:Exons / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D009447:Neuroblastoma / Q000473:pathology; D009474:Neurons / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D020543:Proteome; D034622:RNA Interference; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D034702:RNA-Binding Protein FUS / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D016415:Sequence Alignment; D017385:Sequence Homology; D059467:Transcriptome
chemical_list: D004268:DNA-Binding Proteins; C000599550:FUS protein, human; D009363:Neoplasm Proteins; D020033:Protein Isoforms; D020543:Proteome; D012322:RNA Precursors; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D034741:RNA, Small Interfering; D034702:RNA-Binding Protein FUS; C000598624:TARDBP protein, human
keywords: ALS; Exon Array; FUS; RNA-binding protein; Splicing; TDP-43