Biology subjects

Vojtesek, B.

Publications and source records attributed to Vojtesek, B..

4 recordsLinked to original sources

bioRxiv2026

Cancer-associated synonymous mutations reveal stress signal-dependent mRNA folding that selectively modulates protein function.

Recent technical advances have facilitated studies on changes in mRNA structures in response to signaling pathways. However, if mRNA structures can affect the function of the encoded protein remains poorly understood. In-cell RNA structural probing (SHAPE-MaP) demonstrates how two cancer-associated synonymous mutations (CASMs) at proline codon 34 (c.102 C>A and c.102 C>G) prevent DNA damage-induced TP53 mRNA folding, whereas the non-cancer-associated c.102C>U mutation does not. Transcript and chromatin immunoprecipitation (ChIP) analysis reveal that p53 expressed from CASM34 has reduced promoter binding and reduced induction of p53 downstream target genes PUMA and 14-3-3-{sigma}, but not p21CDKN1A. Transcriptome analysis reveals a CASM34-mediated global attenuation of DNA damage-responsive gene expression. Together, the results demonstrate that CASM34 interferes with signal-induced p53 mRNA folding during DNA damage, leading to selective modulation of protein activity. More broadly, our findings highlight a general concept by which cancer-associated synonymous mutations target signal-induced mRNA structures that influence the encoded protein. Significance statementRecent studies have revealed that synonymous mutations can target RNA metabolism and are associated with numerous diseases; however, how these mutations affect the function of the encoded protein remain unclear. Using in-cell RNA structural probing, we show that two cancer-associated synonymous mutations in TP53 disrupt DNA damage-induced folding of p53 mRNA. These mutations selectively impair p53 promoter binding and transcriptional activation of specific downstream target genes. Our results demonstrate that disruption of signal-induced mRNA folding by synonymous mutations can modulate the activity of the encoded protein. More broadly, this work identifies signal-responsive mRNA structures as functional targets of cancer-associated synonymous mutations.

molecular biology↗

bioRxiv2022

MDM2 inhibitors, nutlin-3a and navtemadelin, retain efficacy in human and mouse cancer cells cultured in hypoxia

Activation of p53 by small molecule MDM2 inhibitors can induce cell cycle arrest or death in p53 wildtype cancer cells. However, cancer cells exposed to hypoxia can develop resistance to other small molecules, such as chemotherapies, that activate p53. Here, we evaluated whether hypoxia could render cancer cells insensitive to two MDM2 inhibitors with different potencies, nutlin-3a and navtemadlin. Inhibitor efficacy and potency were evaluated under short-term hypoxic conditions in human and mouse cancer cells expressing different p53 genotypes (wild-type, mutant, or null). Treatment of wild-type p53 cancer cells with MDM2 inhibitors reduced cell growth by >75% in hypoxia through activation of the p53-p21 signaling pathway; no inhibitor-induced growth reduction was observed in hypoxic mutant or null p53 cells except at very high concentrations. The concentration of inhibitors needed to induce the maximal p53 response was not significantly different in hypoxia compared to normoxia. However, inhibitor efficacy varied by species and by cell line, with stronger effects at lower concentrations observed in human cell lines than in mouse cell lines grown as 2D and 3D cultures. Together, these results indicate that MDM2 inhibitors retain efficacy in hypoxia, suggesting they could be useful for targeting acutely hypoxic cancer cells.

cancer biology↗

bioRxiv2022

SRSF1 as a novel interacting partner for IFITM1/3 unravels the emergent role of IFITM1/3 mediating protein expression

IFITM proteins play a role in cancer cell progression through undefined mechanisms. Here, we propose an emergent role of IFITM1/3 regulating protein synthesis. SBP-tagged IFITM1 protein was used to identify an association of IFITM1 protein with the cytosolic isoform of SRSF1 that transports mRNA to the ribosome. This cytosolic association was confirmed in situ using proximity ligation assays for SRSF1 and IFITM1/3, suggesting a role associated with translation. Accordingly, IFITM1/3 were shown to interact with HLA-B mRNA in response to IFN{gamma} stimulation using RNA-protein proximity ligation assays. In addition, shotgun RNA sequencing in IFITM1/IFITM3 null cells and wt-SiHa cells indicated that reduced HLA-B gene expression does not account for lowered HLA-B protein synthesis in response to IFN{gamma}. Furthermore, ribosome profiling using sucrose gradient sedimentation identified a reduction in 80S ribosomal fraction an IFITM1/IFITM3 null cells compared to their wild-type counterpart, partially reverted by IFITM1/3 complementation. Our data all together link the binding of IFITM1/3 proteins to HLA-B mRNA and SRSF1 as a mechanism to catalyze the synthesis of target proteins, suggesting an RNA chaperonin role for IFITM1/3 proteins. SignificanceIFITMs are widely studied for their role in inhibiting viruses, and multiple studies have associated IFITMs with cancer progression. However, mechanistic insights are not well understood. Our study proposes that IFITMs have a role regulating protein synthesis, a pivotal function highly relevant for viral infection and cancer progression. Our results suggest that IFITM1/3 is present in the ribosomal fraction and regulates particular protein expression; among them, we identified HLA-B. Changes in HLA-B expression could impact the presentation and recognition of oncogenic antigens on the cell surface by cytotoxic T cells and, ultimately, limit tumor cell eradication. In addition, the role of IFITMs mediating protein translation is relevant, as has the potential of regulating the expression of viral and oncogenic proteins.

cancer biology↗

bioRxiv2021

A spindle and thread-mechanism unblocks translation of N-terminally disordered proteins

Protein disorder is a major hurdle for structural biology. A prominent example is the tumour suppressor p53, whose low expression levels and poor conformational stability due to a high degree of disorder pose major challenges to the development of cancer therapeutics. Here, we address these issues by fusing p53 to an engineered spider silk domain termed NT*. The chimeric protein displays highly efficient translation in vitro and in E. coli and is fully active in human cancer cells. The transmission electron microscopy structure and native mass spectrometry reveal that the full-length p53 fusion protein adopts a compact conformation. Molecular dynamics simulations show that the disordered transactivation domain of p53 is wound around the NT* domain via a series of folding events, resulting in a globular structure. We find that expression of B-Raf, another partially disordered cancer target, is similarly enhanced by fusion to NT*. In summary, we demonstrate how inducing co-translational folding via a molecular "spindle and thread" mechanism can overcome poor translation efficiency of partially disordered proteins.

biochemistry↗