Multiple cellular processes are regulated by scaffold proteins that serve as platforms for the formation of protein complexes, regulating their spatial and temporal organization. Impaired expression and mutations in scaffold-encoding genes lead to alterations in the cellular process associated with pathological states.
ITSN1 is a scaffold that modulates clathrin-mediated endocytosis, vesicular transport, actin cytoskeleton rearrangement, and several mitogenic signaling pathways. Altered ITSN1 expression is associated with the proliferation and invasion of glioblastoma, neuroblastoma, and lung cancer cells, as well as with the development of Down syndrome, Alzheimer’s disease, and Huntington’s disease.
Recently, it has been found that in addition to the cytoplasm, ITSN1 also localizes in a cell nucleus, though the functional significance of this phenomenon remains unknown. The current work concerns the analysis of the putative role of ITSN1 in a cell nucleus and the interaction between ITSN1 and nuclear RNA-binding proteins.
Using fluorescent microscopy, we found that endogenous and overexpressed GFP-labeled ITSN1 localized in HeLa cells nuclei. Analyzing the protein interaction databases, we identified thirty-nine putative ITSN1 partners, that are involved in the regulation of mRNA processing, chromatin remodeling, DNA reparation, and replication. Using the GST pull-down approach, we confirmed the interaction between ITSN1 and RNA-binding proteins SAM68, LARP6, and WBP11 in vitro.
Considering the functional features of SAM68, in particular, its role in the oncogenic transformation, we performed the analysis of the interaction between ITSN1 and SAM68. SAM68 is an RNA-binding protein mainly involved in mRNA processing, specifically alternative splicing. The protein stimulates the formation of pro-oncogenic isoforms, whereas, in neurons, it induces the expression of neuron-specific transcripts. We demonstrated that ITSN1 and SAM68 colocalized in HeLa cells nuclei and cytoplasm, whereas the interaction between proteins was mediated by the SH3A domain of ITSN1 and the N-terminal proline motif P0 of SAM68.
Similar to other RNA-binding proteins, SAM68 contains RG-enriched disordered regions prone to aggregation. We demonstrated that ITSN1 suppresses SAM68 aggregation in vitro possibly by masking RG-motifs located in close proximity to the P0 proline motif. For the first time, we identified that ITSN1 directly interacts with nucleic acids, in particular, RNA and single-stranded DNA. The interaction was confirmed by several approaches, including the mRNA mobility gel shift assay and atomic force microscopy. The detected interaction is mediated by the fourth SH3 domain (SH3D) of ITSN1. Using NMR spectrometry, we identified several positively-charged and hydrophobic residues, that form the putative nucleic acid-binding site on the surface of ITSN1 SH3D domain. We proposed that the interaction could stabilize the formation of the complexes between ITSN1 and RNA-binding proteins.
It has been previously shown that SAM68 forms specific nuclear bodies in HeLa cells with unknown function. Here, we demonstrated that ITSN1 depletion stimulated the accumulation of SAM68 in nuclear bodies in HeLa cells. Moreover, accumulation of overexpressed ITSN1 in the nucleus stimulated the dissociation of SAM68 nuclear bodies while deletion of SH3A domain did not affect SAM68 nuclear bodies formation, which allowed us to suggest that ITSN1 could prevent SAM68 aggregation in a nucleus.
It is known that SAM68 regulates alternative splicing of pre-mRNA transcripts of several genes involved in carcinogenesis. These genes include BCL-X, CD44, AR-V, mTOR, SRSF1, CCND1, BIRC5, etc. In the present work, the effect of ITSN1 knockdown on the SAM68-mediated alternative splicing events in HeLa cells was analyzed. ITSN1 knockdown caused a two-fold increase in the expression of the pro-oncogenic isoform of splicing factor SRSF1. Overexpression of this SRSF1 isoform is known to activate the epithelial-mesenchymal transition in various cancer cells. As SAM68 is one of the regulators of the expression and alternative splicing of SRSF1 pre-mRNA, the obtained results indicated that the suppression of ITSN1 may increase the functional activity of SAM68, in particular, in the nuclear bodies.
As a result, we proposed a hypothetical model of the functional interplay between ITSN1 and SAM68 in a nucleus. According to the model, protein ITSN1 directly affects the aggregation of SAM68 in a cell nucleus. As ITSN1 directly binds mRNA, the formation of a triple complex in a cell nucleus prevents SAM68 assembly and induces the dissociation of SAM68 nuclear bodies. Hence, ITSN1 regulates the functioning of SAM68 contributing to the processes of SAM68-mediated alternative splicing, associated with the expression of specific isoforms involved in carcinogenesis, in particular, SRSF1.
