A multi-faceted approach to unravel coding and non-coding gene fusions and target chimeric proteins in ataxia.

Ataxia represents a heterogeneous group of neurodegenerative disorders characterized by a loss of balance and coordination, often resulting from mutations in genes vital for cerebellar function and maintenance. Recent advances in genomics have identified gene fusion events as critical contributors to various cancers and neurodegenerative diseases. However, their role in ataxia pathogenesis remains largely unexplored. Our study Hdelved into this possibility by analyzing RNA sequencing data from 1443 diverse samples, including cell and mouse models, patient samples, and healthy controls. We identified 7067 novel gene fusions, potentially pivotal in disease onset. These fusions, notably in-frame, could produce chimeric proteins, disrupt gene regulation, or introduce new functions. We observed conservation of specific amino acids at fusion breakpoints and identified potential aggregate formations in fusion proteins, known to contribute to ataxia. Through AI-based protein structure prediction, we identified topological changes in three high-confidence fusion proteins-TEN1-ACOX1, PEX14-NMNAT1, and ITPR1-GRID2-which could potentially alter their functions. Subsequent virtual drug screening identified several molecules and peptides with high-affinity binding to fusion sites. Molecular dynamics simulations confirmed the stability of these protein-ligand complexes at fusion breakpoints. Additionally, we explored the role of non-coding RNA fusions as miRNA sponges. One such fusion, RP11-547P4-FLJ33910, showed strong interaction with hsa-miR-504-5p, potentially acting as its sponge. This interaction correlated with the upregulation of hsa-miR-504-5p target genes, some previously linked to ataxia. In conclusion, our study unveils new aspects of gene fusions in ataxia, suggesting their significant role in pathogenesis and opening avenues for targeted therapeutic interventions.Communicated by Ramaswamy H. Sarma.

Proteomics analysis identifies the ribosome associated coiled-coil domain-containing protein-124 as a novel interaction partner of nucleophosmin-1.

BACKGROUND INFORMATION: Coiled-coil domain-containing protein-124 (Ccdc124) is a conserved eukaryotic ribosome-associated RNA-binding protein which is involved in resuming ribosome activity after stress-related translational shutdown. Ccdc124 protein is also detected at cellular localizations devoid of ribosomes, such as the centrosome, or the cytokinetic midbody, but its translation-independent cellular function is currently unknown. RESULTS: By using an unbiased LC-MS/MS-based proteomics approach in human embryonic kidney (HEK293) cells, we identified novel Ccdc124 partners and mapped the cellular organization of interacting proteins, a subset of which are known to be involved in nucleoli biogenesis and function. We then identified a novel interaction between the cancer-associated multifunctional nucleolar marker nucleophosmin (Npm1) and Ccdc124, and we characterized this interaction both in HEK293 (human embryonic kidney) and U2OS (osteosarcoma) cells. As expected, in both types of cells, Npm1 and Ccdc124 proteins colocalized within the nucleolus when assayed by immunocytochemical methods, or by monitoring the localization of green fluorescent protein-tagged Ccdc124. CONCLUSIONS: The nucleolar localization of Ccdc124 was impaired when Npm1 translocates from the nucleolus to the nucleoplasm in response to treatment with the DNA-intercalator and Topo2 inhibitor chemotherapeutic drug doxorubicin. Npm1 is critically involved in maintaining genomic stability by mediating various DNA-repair pathways, and over-expression of Npm1 or specific NPM1 mutations have been previously associated with proliferative diseases, such as acute myelogenous leukemia, anaplastic large-cell lymphoma, and solid cancers originating from different tissues. SIGNIFICANCE: Identification of Ccdc124 as a novel interaction partner of Nmp1 within the frame of molecular mechanisms involving nucleolar stress-sensing and DNA-damage response is expected to provide novel insights into the biology of cancers associated with aberrations in NPM1.

Identification of novel TUBB1 variants in patients with macrothrombocytopenia.

Background/aim: Macrothrombocytopenia is an autosomal-dominant disorder characterized by increased platelet size and a decreased number of circulating platelets. The membrane skeleton and the link between actin filaments of the skeleton and microtubules, which consist of alpha and beta tubulin [including the tubulin beta-1 chain (TUBB1)] heterodimers, are important for normal platelet morphology, and defects in these systems are associated with macrothrombocytopenia. Materials and methods: In this study, we sequenced the exons of the TUBB1 gene using DNA isolated from the peripheral blood samples of healthy controls (n = 47) and patients with macrothrombocytopenia (n = 37) from Turkey. The TUBB1 expression levels in fractioned blood samples from patients and healthy controls were analyzed by RT-qPCR and Western blot. Microtubule organization of the platelets in the peripheral blood smears of patients, and in mutant TUBB1-transfected HeLa cells, were analyzed by immunofluorescence staining. Results: A new TUBB1 c.803G>T (p.T178T) variant was detected in all of the control and patient samples. Importantly, we found 3 new heterozygous TUBB1 variants predicting amino acid substitutions: G146R (in 1 patient), E123Q (in 1 patient), and T274M (in 4 patients); the latter variant was associated with milder thrombocytopenia in cancer patients treated with paclitaxel. Ectopic expression of TUBB1 T274M/R307H variant in HeLa cells resulted in irregular microtubule organization. Conclusion: Further clinical and functional studies of the newly identified TUBB1 variants may offer important insights into their pathogenicity in macrothrombocytopenia.

The Thyroid Na+/I- Symporter: Molecular Characterization and Genomic Regulation.

Iodide (I-) is an essential constituent of the thyroid hormones triiodothyronine (T3) and thyroxine (T4), and the iodide concentrating mechanism of the thyroid gland is essential for the synthesis of these hormones. In addition, differential uptake of iodine isotopes (radioiodine) is a key modality for the diagnosis and therapy of thyroid cancer. The sodium dependent iodide transport activity of the thyroid gland is mainly attributed to the functional expression of the Na+/I- Symporter (NIS) localized at the basolateral membrane of thyrocytes. In this paper, we review and summarize current data on molecular characterization, on structure and function of NIS protein, as well as on the transcriptional regulation of NIS encoding gene in the thyroid gland. We also propose that a better and more precise understanding of NIS gene regulation at the molecular level in both healthy and malignant thyroid cells may lead to the identification of small molecule candidates. These could then be translated into clinical practice for better induction and more effective modulation of radioiodine uptake in dedifferentiated thyroid cancer cells and in their distant metastatic lesions.

Na+/I- symporter and type 3 iodothyronine deiodinase gene expression in amniotic membrane and placenta and its relationship to maternal thyroid hormones.

Placental type 3 iodothyronine deiodinase (D3) potentially protects the fetus from the elevated maternal thyroid hormones. Na(+)/I(-) symporter (NIS) is a plasma membrane glycoprotein, which mediates active iodide uptake. Our objectives were to establish the distribution of NIS and D3 gene expressions in the placenta and the amniotic membrane and to investigate the relationship between placental D3 and NIS gene expressions and maternal iodine, selenium, and thyroid hormone status. Thyroid hormones, urinary iodine concentration (UIC), and selenium levels were measured in 49 healthy term pregnant women. NIS and D3 gene expressions were studied with the total mRNA RT-PCR method in tissues from maternal placenta (n = 49), fetal placenta (n = 9), and amniotic membrane (n = 9). NIS and D3 gene expressions were shown in the fetal and maternal sides of the placenta and amniotic membrane. Mean blood selenium level was 66 ± 26.5 µg/l, and median UIC was 143 µg/l. We could not demonstrate any statistically significant relationship of spot UIC and blood selenium with NIS and D3 expression (p > 0.05). Positive correlations were found between NIS and thyroxine-binding globulin (TBG) (r = 0.3, p = 0.042) and between D3 and preoperative glucose levels (r = 0.4, p = 0.006). D3 and NIS genes are expressed in term placenta and amniotic membrane; thus, in addition to placenta, amniotic membrane contributes to regulation of maternofetal iodine and thyroid hormone transmission. Further studies are needed to clarify the relationship between maternal glucose levels and placental D3 expression and between TBG and placental NIS expression.

CD81 interacts with the T cell receptor to suppress signaling.

CD81 (TAPA-1) is a ubiquitously expressed tetraspanin protein identified as a component of the B lymphocyte receptor (BCR) and as a receptor for the Hepatitis C Virus. In an effort to identify trans-membrane proteins that interact with the T-cell antigen receptor (TCR), we performed a membrane yeast two hybrid screen and identified CD81 as an interactor of the CD3delta subunit of the TCR. We found that in the absence of CD81, in thymocytes from knockout mice, TCR engagement resulted in stronger signals. These results were recapitulated in T cell lines that express low levels of CD81 through shRNA mediated silencing. Increased signaling did not result from alterations in the levels of TCR on the surface of T lymphocytes. Although CD81 is not essential for normal T lymphocyte development, it plays an important role in regulating TCR and possibly pre-TCR signal transduction by controlling the strength of signaling. CD81 dependent alterations in thymocyte signaling are evident in increased CD5 expression on CD81 deficient double positive (DP) thymocytes. We conclude that CD81 interacts with the T cell receptor to suppress signaling.