Project description:Here we utilized an unbiased whole RNA-seq approach to characterize the transcriptional effects of YB-1 knockdown in three mesothelioma cell lines (MSTO, VMC23 and REN cells). Bioinformatic analysis showed that YB-1 knockdown regulated 150 common genes enriched for mitotic genes, integrins and extracellular matrix. However, each cell line also displayed a unique transcriptional signature, with differential deregulation of cell death, the cell cycle, and p53 signaling observed across the three cell lines.
Project description:We present results of RNA-Seq, Ribo-Seq, and RIP-Seq (YB-1, YB-3) experiments performed in HEK293T cells, as well as in HEK293T cells with YB-1 knockout and overexpression. The data shows YB-1 function as a global translation inhibitor and YB-3 ability to substitute YB-1 in its function in YB-1 knockout mutant.
Project description:To understand the mechanistic basis of YB-1’s regulation of mRNA splicing in response to DNA damage in Multiple Myeloma, we performed RNA immunoprecipitation (RIP) and sequencing of ILF2-bound transcripts under both physiological and DNA damage (melphalan treatment) conditions. Cells were treated with melphalan for 10 hours. (RIP) and sequencing of YB-1-bound RNAs was performed in the JJN3 line (two biological replicates/condition).
Project description:Outcomes for metastatic bone sarcomas, Ewing sarcoma and osteosarcoma, are dismal and remain unchanged for decades. Oxidative stress attenuates melanoma metastasis, and melanoma cells must reduce oxidative stress to metastasize. To explore this in sarcomas, we screened libraries of approved compounds for agents sensitizing sarcoma cells to oxidative stress. This identified the class I HDAC inhibitor MS-275 as enhancing sensitivity to reactive oxygen species (ROS). Mechanistically, MS-275 inhibits YB-1 deacetylation, decreasing physical binding between YB-1 and the 5UTR of NFE2L2, thereby non-transcriptionally reducing translation and expression of the master antioxidant factor, NRF2, which reduces cellular ROS. Indeed, global acetylomics revealed that MS-275 promotes rapid acetylation of the YB-1 RNA binding protein at lysine-81, blocking RNA binding and translational activation of NFE2L2, encoding NRF2, as well as known YB-1 mRNA targets, HIF1A and the stress granule nucleator, G3BP1. MS-275 dramatically reduced sarcoma metastasis in vivo, but an MS-275-resistant YB-1 K81-to-alanine (K81A) mutant restored metastatic capacity and NRF2, HIF1α, and G3BP1 synthesis in MS-275 treated mice. These studies describe a novel function for MS-275 through enhanced YB-1 acetylation, thus inhibiting YB-1 translational control of key cytoprotective factors and its pro-metastatic activity.
Project description:In this study, we generated transcriptome profiles from HEK293T, HEK293T ΔYB-1ΔYB-3, and HEK293T ΔYB-1ΔYB-3 with the restored expression of YB-1 and/or YB-3. The data are used to explore YB-1 and YB-3 influence on transcriptome and translatome and their functional interchangeability in transcription and translation.
Project description:Female sterile (1) Yb (Yb) is a primary component of Yb bodies, perinuclear foci known as the site of PIWI-interacting RNA (piRNA) biogenesis in Drosophila ovarian somatic cells. Yb consists of Helicase C-terminal (Hel-C), RNA helicase and extended Tudor (eTud) domains. We previously showed that the RNA helicase domain is necessary for Yb−RNA interaction, Yb body formation and piRNA biogenesis. Here, we investigated the functions of the two other domains and found that Hel-C and eTud are necessary for Yb to self-associate and to interact with RNAs and other Yb body components, respectively. Both domains were essential for Yb body formation and transposon silencing. Without eTud, piRNA production was completely impaired. Loss of Hel-C severely reduced the levels of transposon-targeting piRNAs, although genic piRNAs unrelated to transposon silencing were still produced. Similar phenotypes were observed when mutations in flamenco, the primary source of transposon-targeting piRNAs, led to the expression of attenuated RNAs. Yb bodies are liquid-like multivalent condensates whose assembly depends on Yb self-association and widespread Yb−flamenco RNA binding.
Project description:Background: Malignant mesothelioma is an aggressive cancer with poor prognosis. It is characterized by prominent extracellular matrix, mesenchymal tumor cell phenotypes and chemoresistance. In this study, the ability of pirfenidone to alter mesothelioma cell proliferation and migration as well as mesothelioma tumor microenvironment was evaluated. Pirfenidone is an anti-fibrotic drug used in the treatment of idiopathic pulmonary fibrosis and has also anti-proliferative activities. Aims: Transcriptional profiling using RNA sequencing of human mesothelioma xenograft tumors treated with PBS or pirfenidone. Aim was to compare extracellular matrix and fibrosis related genes. Results: Treatment of mice harboring mesothelioma xenografts with pirfenidone alone did not reduce tumor proliferation in vivo. However, pirfenidone modified the tumor microenvironment by reducing the expression of extracellular matrix associated genes. In addition, GREM1 expression was downregulated by pirfenidone in vivo. Conclusion: By reducing two major upregulated pathways in mesothelioma and by targeting tumor cells and the microenvironment pirfenidone may present a novel anti-fibrotic and anti-cancer adjuvant therapy for mesothelioma.
Project description:piRNAs direct Piwi to repress transposons to maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) concentrate into perinuclear foci adjacent to Yb bodies, termed Flam bodies. Although flam expression is not required for Yb body formation, Yb, the core component of Yb bodies, is required for Flam body formation. Abolishment of the RNA-binding activity of Yb disrupts both Yb bodies and Flam bodies. Loss of Zucchini, an endoribonuclease necessary for piRNA maturation, enlarges Flam bodies, which now superimpose with Yb bodies. Yb directly binds flam, but not neighboring protein-coding gene, transcripts. Thus, Yb integrates piRNA processing factors and piRNA intermediates into Yb bodies and Flam bodies, respectively, through direct binding to enhance piRNA biogenesis and formation of piRNA-inducing silencing complexes.