TDP-43 AND NOVA-1 RNA BINDING PROTEINS AS SPLICING REGULATORS OF TNIK GENE, A SCHIZOPHRENIA GENETIC RISK FACTOR
Tesi di Dottorato
Data di Pubblicazione:
2019
Citazione:
TDP-43 AND NOVA-1 RNA BINDING PROTEINS AS SPLICING REGULATORS OF TNIK GENE, A SCHIZOPHRENIA GENETIC RISK FACTOR / V. Gumina ; tutor: A. Ratti ; coordinatore: R. Ghidoni. DIPARTIMENTO DI FISIOPATOLOGIA MEDICO-CHIRURGICA E DEI TRAPIANTI, 2019 Jan 17. 31. ciclo, Anno Accademico 2018. [10.13130/gumina-valentina_phd2019-01-17].
Abstract:
TDP-43, a RNA-binding protein (RBP) involved in different steps of RNA metabolism, forms pathological aggregates in affected tissues of the majority of amyotrophic lateral sclerosis (ALS) patients and of a subset of frontotemporal dementia (FTLD) cases. Our group has recently demonstrated that TDP-43 regulates the alternative splicing of several pre-mRNAs related to neuronal metabolism, including TNIK, encoding for a Ser/Thr kinase highly expressed in the brain. TDP-43 promotes the skipping of TNIK alternative exon 15, which encodes for a 29-amino-acid sequence in a region of the protein with unknown function. The TNIK gene was found to be genetically associated to psychiatric disorders, such as schizophrenia, as a risk factor, and to intellectual disability for null-mutations. In neurons, TNIK is involved in regulating different processes, including synapse formation, dendrite arborization and neurogenesis.
In this study, our first aim was to investigate the regulation of TNIK exon 15 alternative splicing during neuronal differentiation in order to better characterize the role played by the ubiquitously expressed and ALS/FTLD-associated TDP-43 splicing factor in a neuronal context. Our second aim was to investigate if the specific expression of TNIK exon15-cointaining (TNIKex15) protein isoforms may be important to maintain TNIK physiological function in neurons, mainly focusing on its activity in neurite development.
We first observed that TNIK exon 15 alternative splicing was differently regulated in human adult tissues and TNIKex15 isoforms were exclusively expressed in brain, spinal cord and skeletal muscle. Given the prevalent expression of TNIKex15 isoforms in the central nervous system, we further investigated this alternative splicing event in in vitro models to evaluate its regulation during the neuronal differentiation process. We found a significant increase of TNIKex15 transcripts in both SK-N-BE cells treated with retinoic acid and in human iPSCs differentiated into neurons. Moreover, TNIKex15 protein isoforms were specifically expressed in neuron-differentiated cells showing a prevalent perinuclear distribution in immunofluorescence analyses.
Since we previously showed that TNIK exon 15 inclusion increases upon TDP-43 knock-down, we measured TDP-43 protein content during neuronal differentiation in vitro but we found no changes. We therefore investigated the possible involvement of the neuron-specific splicing factor NOVA-1, specifically expressed in our neuron-differentiated cells, in regulating TNIK processing. In HEK293T cells, NOVA-1 over-expression increased TNIK exon 15 inclusion without negatively affecting TDP-43 protein content compared to mock-transfected cells. Furthermore, by minigene splicing assays we evaluated the NOVA-1 interplay with TDP-43 in regulating TNIK exon 15 splicing. In this analysis, we also included hnRNPA2/B1, an ubiquitous RBP that co-operates with TDP-43 in regulating its splicing activity and that we found to promote TNIK exon 15 skipping, similarly to TDP-43. In competition assay with TDP-43 and hnRNPA2/B1, NOVA-1 completely abrogated their exon skipping activity on TNIK gene interacting with TDP-43 and hnRNPA2/B1 proteins in a RNA-dependent manner.
As our results suggested a neuronal relevance for TNIKex15 protein isoforms, we further investigated the specific function of these isoforms in neurite development in murine primary cortical neurons. TNIKex15 over-expression negatively affected neurite development, reducing neurite number, as already describe in literature. When we analyzed TNIKex15-expressing neurons, we observed reduced filopodia number at growth cones, reduced soma area and filamentous actin (F-actin) levels compared to control cells. In contrast, upon TNIK ex
Tipologia IRIS:
Tesi di dottorato
Keywords:
TDP-43; NOVA-1; TNIK; splicing; ALS; schizophrenia
Elenco autori:
V. Gumina
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