SRSF1 interactome determined by proximity labeling reveals direct interaction with spliceosomal RNA helicase DDX23.

SRSF1 is the founding member of the SR protein family. It is required-interchangeably with other SR proteins-for pre-mRNA splicing in vitro, and it regulates various alternative splicing events. Dysregulation of SRSF1 expression contributes to cancer and other pathologies. Here, we characterized SRSF1's interactome using proximity labeling and mass spectrometry. This approach yielded 190 proteins enriched in the SRSF1 samples, independently of the N- or C-terminal location of the biotin-labeling domain. The detected proteins reflect established functions of SRSF1 in pre-mRNA splicing and reveal additional connections to spliceosome proteins, in addition to other recently identified functions. We validated a robust interaction with the spliceosomal RNA helicase DDX23/PRP28 using bimolecular fluorescence complementation and in vitro binding assays. The interaction is mediated by the N-terminal RS-like domain of DDX23 and both RRM1 and the RS domain of SRSF1. During pre-mRNA splicing, DDX23's ATPase activity is essential for the pre-B to B spliceosome complex transition and for release of U1 snRNP from the 5' splice site. We show that the RS-like region of DDX23's N-terminal domain is important for spliceosome incorporation, while larger deletions in this domain alter subnuclear localization. We discuss how the identified interaction of DDX23 with SRSF1 and other SR proteins may be involved in the regulation of these processes.

p160 nuclear receptor coactivator family members and their role in rare fusion­driven neoplasms (Review).

Gene fusions with translocations involving nuclear receptor coactivators (NCoAs) are relatively common among fusion-driven malignancies. NCoAs are essential mediators of environmental cues and can modulate the transcription of downstream target genes upon binding to activated nuclear receptors. Therefore, fusion proteins containing NCoAs can become strong oncogenic drivers, affecting the cell transcriptional profile. These tumors show a strong dependency on the fusion oncogene; therefore, the direct pharmacological targeting of the fusion protein becomes an attractive strategy for therapy. Currently, different combinations of chemotherapy regimens are used to treat a variety of NCoA-fusion-driven tumors, but given the frequent tumor reoccurrence, more efficient treatment strategies are needed. Specific approaches directed towards inhibition or silencing of the fusion gene need to be developed while minimizing the interference with the original genes. This review highlights the relevant literature describing the normal function and structure of NCoAs and their oncogenic activity in NCoA-gene fusion-driven cancers, and explores potential strategies that could be effective in targeting these fusions.

Patient-derived xenografts and in vitro model show rationale for imatinib mesylate repurposing in HEY1-NCoA2-driven mesenchymal chondrosarcoma.

Mesenchymal chondrosarcoma (MCS) is a high-grade malignancy that represents 2-9% of chondrosarcomas and mostly affects children and young adults. HEY1-NCoA2 gene fusion is considered to be a driver of tumorigenesis and it has been identified in 80% of MCS tumors. The shortage of MCS samples and biological models creates a challenge for the development of effective therapeutic strategies to improve the low survival rate of MCS patients. Previous molecular studies using immunohistochemical staining of patient samples suggest that activation of PDGFR signaling could be involved in MCS tumorigenesis. This work presents the development of two independent in vitro and in vivo models of HEY1-NCoA2-driven MCS and their application in a drug repurposing strategy. The in vitro model was characterized by RNA sequencing at the single-cell level and successfully recapitulated relevant MCS features. Imatinib, as well as specific inhibitors of ABL and PDGFR, demonstrated a highly selective cytotoxic effect targeting the HEY1-NCoA2 fusion-driven cellular model. In addition, patient-derived xenograft (PDX) models of MCS harboring the HEY1-NCoA2 fusion were developed from a primary tumor and its distant metastasis. In concordance with in vitro observations, imatinib was able to significantly reduce tumor growth in MCS-PDX models. The conclusions of this study serve as preclinical results to revisit the clinical efficacy of imatinib in the treatment of HEY1-NCoA2-driven MCS.

OH1 from Orf Virus: A New Tyrosine Phosphatase that Displays Distinct Structural Features and Triple Substrate Specificity.

Viral tyrosine phosphatases such as VH1 from Vaccinia and Variola virus are recognized as important effectors of host-pathogen interactions. While proteins sharing sequence to VH1 have been identified in other viruses, their structural and functional characterization is not known. In this work, we determined the crystal structure of the VH1 homolog in the Orf virus, herein named OH1. Similarly to Variola and Vaccinia VH1, the structure of OH1 shows a dimer with the typical dual-specificity phosphatase fold. In contrast to VH1, the OH1 dimer is covalently stabilized by a disulfide bond involving residue Cys15 in the N-terminal helix alpha-1 of both monomers, and Cys15 is a conserved residue within the Parapoxvirus genus. The in vitro functional characterization confirms that OH1 is a dual-specificity phosphatase and reveals its ability to dephosphorylate phosphatidylinositol 3,5-bisphosphate, a new activity potentially relevant in phosphoinositide recycling during virion maturation.