| 1. |
- Dass, Randall A., et al.
(författare)
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Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I
- 2016
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Ingår i: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 12:8
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.
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| 2. |
- Mínguez‐Viñas, Teresa, et al.
(författare)
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Two epilepsy‐associated variants in KCNA2 (KV1.2) at position H310 oppositely affect channel functional expression
- 2023
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Ingår i: Journal of Physiology. - : WILEY. - 0022-3751 .- 1469-7793. ; 601:23, s. 5367-5389
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Tidskriftsartikel (refereegranskat)abstract
- Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV-channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV1.2 functional expression. The p.H310Y variant produced ‘dual gain of function’, increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished ‘ball and chain’ inactivation of KV1.2 by KVβ1 subunits, enhancing gain of function. In contrast, p.H310R caused ‘dual loss of function’, diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV-channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis.
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| 3. |
- Prakash, Varsha
(författare)
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Not just protein factories : role of ribosome biogenesis as an architect of epithelial-mesenchymal transition and breast cancer progression
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- RNA polymerase I (Pol I)-mediated transcription of ribosomal DNA (rDNA) is considered to be the rate-limiting step in ribosome biogenesis and is a well-known hallmark of cell growth and proliferation. The process of synthesizing new ribosomes is executed by the coordination of multiple complex processes in the nucleolus. The initial step of transcribing 47S ribosomal RNA (rRNA) transcript by the Pol I complex is followed by its processing into 28S, 18S and 5.8S rRNAs. These transcripts, together with 5S rRNA transcribed by Pol III and auxiliary proteins transcribed by Pol II, proceed to form a mature ribosome after being exported into the cytoplasm. Regulation of ribosome biogenesis occurs in a cell cycle dependent manner, and actively transcribing nucleolar organizing regions (NORS) indicating active rDNA transcription have been associated with tumor proliferation and poor prognosis in cancer patients. Numerous oncogenic and tumor suppressive pathways modulate tumor growth through rDNA transcription. We have previously shown that the tumor suppressive effects of Wnt5a is mediated though suppression of rDNA transcription by recruitment of Dishevelled 1 (DVL1) to the nucleolus and the rDNA gene cassette. In this thesis, we show that de novo ribosome biogenesis is essential for the epithelial-to-mesenchymal transition (EMT), which is indispensable for embryonic development and for the acquisition of migratory phenotype during cancer progression. The induced de novo rRNA synthesis occurring in the absence of cell proliferation is mediated by increased recruitment of Pol I complex components and EMT transcription factor Snail1 to the rDNA gene cassette. This is accompanied by the opening of the otherwise silenced rDNA operons by the release of TTF-I interacting protein 5 (TIP5), a major component of the repressive nuclear chromatin remodeling NoRC complex, from the rDNA. Pharmacological inhibition of rRNA synthesis by the small molecule CX-5461 reduced the invasive capacity of cells in vitro, which correlated with a decrease in mesenchymal proteins, together confirming an important role of de novo ribosome biogenesis in EMT. In accordance with previous literature that have shown association of the mTORC2 complex with ribosomes, expression of Rictor, a mTORC2 complex component, was found to be induced in the nucleolus during EMT. This association of Rictor was observed to be rRNA dependent. Furthermore, inhibition of ribosome biogenesis significantly reduced the nucleolar expression of Rictor. Mouse models of metastatic breast cancer showed reduced tumor volume upon treatment with CX-5461 and a significant reduction in lung metastasis was observed. Interestingly, CX-5461 treated primary tumors were also more differentiated, as they had increased expression of cytokeratin 8/18, and were also Estrogen Receptor-alpha (ERα) positive and Rictor-negative, which altogether correlates with a less aggressive phenotype in 2 the MMTV-PyMT mouse tumor model. Further investigation into the driving mechanism of EMT by de novo ribosome biogenesis revealed pervasive changes in the translational control of gene expression program during EMT. This translational control during EMT was affected by inhibition of de novo rRNA synthesis by the Pol I assembly inhibitor, CX-5461. Though the transcriptional profiles remained the same, about 1478 genes were differentially expressed in the ribosome protected fragments during EMT. CX-5461 treatment blocked the upregulation of 185 and the downregulation of 179 translationally controlled genes. The expression of the translationally controlled genes post-TGFβ stimulation significantly overlapped with the translationally controlled genes affected by CX-5461. Interestingly, a translational reprogramming of the mTORC1 signaling cascade was observed during EMT, revealed by the downregulation of transcripts with short UTRs. This reprogramming was diminished by the inhibition of Pol I mediated rDNA transcription. These findings collectively provide compelling evidence that the EMT-associated ribosome biogenesis program, and by extension the ribosomes generated by this process, fuel the pro-migratory, pro-invasive gene expression program underpinning EMT and thus the mesenchymal phenotype. They also demonstrate compelling evidence that rRNA biogenesis plays a unique and targetable role in metastatic breast cancer development, progression and metastasis.
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| 4. |
- Prakash, Varsha, et al.
(författare)
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Ribosome biogenesis during cell cycle arrest fuels EMT in development and disease
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ER alpha) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.
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