Musashi1 Contribution to Glioblastoma Development via Regulation of a Network of DNA Replication, Cell Cycle and Division Genes

RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in their levels are often observed in tumors with numerous oncogenic RBPs identified in recent years. Musashi1 (Msi1) is an RBP and stem cell gene that controls the balance between self-renewal and differentiation. High Msi1 levels have been observed in multiple tumors including glioblastoma and are often associated with poor patient outcomes and tumor growth. A comprehensive genomic analysis identified a network of cell cycle/division and DNA replication genes and established these processes as Msi1’s core regulatory functions in glioblastoma. Msi1 controls this gene network via two mechanisms: direct interaction and indirect regulation mediated by the transcription factors E2F2 and E2F8. Moreover, glioblastoma lines with Msi1 knockout (KO) displayed increased sensitivity to cell cycle and DNA replication inhibitors. Our results suggest that a drug combination strategy (Msi1 + cell cycle/DNA replication inhibitors) could be a viable route to treat glioblastoma.

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SAM68: Signal Transduction and RNA Metabolism in Human Cancer

BioMed Research International ◽

10.1155/2015/528954 ◽

2015 ◽

Vol 2015 ◽

pp. 1-14 ◽

Cited By ~ 42

Author(s):

Paola Frisone ◽

Davide Pradella ◽

Anna Di Matteo ◽

Elisa Belloni ◽

Claudia Ghigna ◽

...

Keyword(s):

Cell Cycle ◽

Signal Transduction ◽

Nuclear Export ◽

Cell Biology ◽

Rna Binding ◽

Rna Binding Proteins ◽

Splice Variants ◽

Human Cancer ◽

Rna Metabolism ◽

Regulatory Sequences

Alterations in expression and/or activity of splicing factors as well as mutations incis-acting splicing regulatory sequences contribute to cancer phenotypes. Genome-wide studies have revealed more than 15,000 tumor-associated splice variants derived from genes involved in almost every aspect of cancer cell biology, including proliferation, differentiation, cell cycle control, metabolism, apoptosis, motility, invasion, and angiogenesis. In the past decades, several RNA binding proteins (RBPs) have been implicated in tumorigenesis. SAM68 (SRC associated in mitosis of 68 kDa) belongs to the STAR (signal transduction and activation of RNA metabolism) family of RBPs. SAM68 is involved in several steps of mRNA metabolism, from transcription to alternative splicing and then to nuclear export. Moreover, SAM68 participates in signaling pathways associated with cell response to stimuli, cell cycle transitions, and viral infections. Recent evidence has linked this RBP to the onset and progression of different tumors, highlighting misregulation of SAM68-regulated splicing events as a key step in neoplastic transformation and tumor progression. Here we review recent studies on the role of SAM68 in splicing regulation and we discuss its contribution to aberrant pre-mRNA processing in cancer.

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Widespread FUS mislocalization is a molecular hallmark of ALS

10.1101/491787 ◽

2019 ◽

Cited By ~ 1

Author(s):

Giulia E. Tyzack ◽

Raphaelle Luisier ◽

Doaa M. Taha ◽

Jacob Neeves ◽

Miha Modic ◽

...

Keyword(s):

Motor Neurons ◽

Nuclear Export ◽

Rna Binding ◽

Rna Binding Proteins ◽

Intron Retention ◽

Direct Interaction ◽

Spinal Cord Tissue ◽

Cytoplasmic Inclusions ◽

Sporadic Als ◽

Fus Protein

AbstractAmyotrophic lateral sclerosis (ALS)-causing mutations clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins (RBPs), which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RBPs mislocalize without necessarily becoming constituents of ALS cytoplasmic inclusions themselves remains unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RBP Fused in Sarcoma (FUS), in an unaggregated state, may occur more widely in ALS that previously recognized. To address this hypothesis, we analysed motor neurons (MNs) from an human ALS induced-pluripotent stem cells (iPSC) model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron-retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.

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The RNA-Binding Protein Musashi1 Regulates a Network of Cell Cycle Genes in Group 4 Medulloblastoma

Cells ◽

10.3390/cells11010056 ◽

2021 ◽

Vol 11 (1) ◽

pp. 56

Author(s):

Mirella Baroni ◽

Gabriela D. A. Guardia ◽

Xiufen Lei ◽

Adam Kosti ◽

Mei Qiao ◽

...

Keyword(s):

Cell Cycle ◽

Poor Prognosis ◽

Therapeutic Strategy ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Seq ◽

Molecular Subgroups ◽

Cell Cycle Genes ◽

Group 4 ◽

Genomic Analyses

Medulloblastoma is the most common malignant brain tumor in children. Treatment with surgery, irradiation, and chemotherapy has improved survival in recent years, but patients are frequently left with devastating neurocognitive and other sequelae. Patients in molecular subgroups 3 and 4 still experience a high mortality rate. To identify new pathways contributing to medulloblastoma development and create new routes for therapy, we have been studying oncogenic RNA-binding proteins. We defined Musashi1 (Msi1) as one of the main drivers of medulloblastoma development. The high expression of Msi1 is prevalent in Group 4 and correlates with poor prognosis while its knockdown disrupted cancer-relevant phenotypes. Genomic analyses (RNA-seq and RIP-seq) indicated that cell cycle and division are the main biological categories regulated by Msi1 in Group 4 medulloblastoma. The most prominent Msi1 targets include CDK2, CDK6, CCND1, CDKN2A, and CCNA1. The inhibition of Msi1 with luteolin affected the growth of CHLA-01 and CHLA-01R Group 4 medulloblastoma cells and a synergistic effect was observed when luteolin and the mitosis inhibitor, vincristine, were combined. These findings indicate that a combined therapeutic strategy (Msi1 + cell cycle/division inhibitors) could work as an alternative to treat Group 4 medulloblastoma.

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Dual Knockdown of Musashi RNA-Binding Proteins MSI-1 and MSI-2 Attenuates Putative Cancer Stem Cell Characteristics and Therapy Resistance in Ovarian Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms222111502 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11502

Author(s):

Maria T. Löblein ◽

Isabel Falke ◽

Hans Theodor Eich ◽

Burkhard Greve ◽

Martin Götte ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Ovarian Cancer ◽

Stem Cell ◽

Cancer Stem Cell ◽

Cancer Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Therapy Resistance ◽

Ovarian Cancer Cells

In ovarian cancer, therapy resistance mechanisms complicate cancer cell eradication. Targeting Musashi RNA-binding proteins (MSI) may increase therapeutic efficacy. Database analyses were performed to identify gene expression associations between MSI proteins and key therapy resistance and cancer stem cell (CSC) genes. Then, ovarian cancer cells were subjected to siRNA-based dual knockdown of MSI-1 and MSI-2. CSC and cell cycle gene expression was investigated using quantitative polymerase chain reaction (qPCR), western blots, and flow cytometry. Metabolic activity and chemoresistance were assessed by MTT assay. Clonogenic assays were used to quantify cell survival post-irradiation. Database analyses demonstrated positive associations between MSI proteins and putative CSC markers NOTCH, MYC, and ALDH4A1 and negative associations with NOTCH inhibitor NUMB. MSI-2 expression was negatively associated with the apoptosis regulator p21. MSI-1 and MSI-2 were positively correlated, informing subsequent dual knockdown experiments. After MSI silencing, CSC genes were downregulated, while cell cycle progression was reduced. Metabolic activity was decreased in some cancer cells. Both chemo- and radioresistance were reduced after dual knockdown, suggesting therapeutic potential. Dual knockdown of MSI proteins is a promising venue to impede tumor growth and sensitize ovarian cancer cells to irradiation and chemotherapy.

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An interaction network of RNA-binding proteins involved in Drosophila oogenesis

10.1101/2020.01.08.899146 ◽

2020 ◽

Author(s):

Prashali Bansal ◽

Johannes Madlung ◽

Kristina Schaaf ◽

Boris Macek ◽

Fulvia Bono

Keyword(s):

Translational Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Direct Interaction ◽

Interaction Network ◽

Splicing Factors ◽

Mrna Regulation ◽

Interaction Partners ◽

Maternal Transcripts

AbstractDuring Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.

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Transformer 2β Regulates the Alternative Splicing of Cell Cycle Regulator Genes to Promote Ovarian Cancer Cell Progression

10.21203/rs.3.rs-853170/v1 ◽

2021 ◽

Author(s):

Peiying Fu ◽

Ting Zhou ◽

Dong Chen ◽

ShiXuan Wang ◽

Ronghua Liu

Keyword(s):

Cell Cycle ◽

Ovarian Cancer ◽

Alternative Splicing ◽

Cancer Progression ◽

Poor Prognosis ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Level

Abstract Background: Late-stage ovarian cancer (OV) has a poor prognosis and a high metastasis rate, but the underlying molecular mechanism is ambiguous. RNA binding proteins (RBPs) play important roles in posttranscriptional regulation in the contexts of neoplasia and tumor metastasis. Results: In this study, we explored the molecular functions of a canonical RBP, TRA2B, in cancer cells. TRA2B knockdown in HeLa cells and whole-transcriptome sequencing (RNA-seq) experiments revealed that the TRA2B-regulated alternative splicing (AS) profile was tightly associated with the mitotic cell cycle, apoptosis, and several cancer pathways. Moreover, hundreds of genes were regulated by TRA2B at the expression level, and their functions were enriched in cell proliferation, cell adhesion and angiogenesis, which are related to cancer progression. We also observed that AS regulation and expression regulation occurred independently by integrating the alternatively spliced and differentially expressed genes. We then explored and validated the carcinogenic functions of TRA2B by knocking down its expression in OV cells. In vivo, a high expression level of TRA2B was associated with a poor prognosis in OV patients. Conclusions: We demonstrated the important roles of TRA2B in ovarian neoplasia and OV progression and identified the underlying molecular mechanisms, facilitating the targeted treatment of OV in the future.

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Human Pumilio proteins directly bind the CCR4-NOT deadenylase complex to regulate the transcriptome

10.1101/2020.11.17.387456 ◽

2020 ◽

Author(s):

Isioma I.I. Enwerem ◽

Nathan D. Elrod ◽

Chung-Te Chang ◽

Ai Lin ◽

Ping Ji ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Molecular Model ◽

Direct Interaction ◽

Vertebrate Development ◽

Regulate Gene Expression ◽

Mrna Decapping ◽

Specific Mrnas ◽

Decapping Enzyme ◽

Pumilio Proteins

AbstractPumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad co-regulation of target mRNAs through the PUM-CNOT repression mechanism.Functional dissection of the domains of PUM1&2 identified a conserved N-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 N-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.

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Human Exonuclease 1 (EXO1) Regulatory Functions in DNA Replication with Putative Roles in Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms20010074 ◽

2018 ◽

Vol 20 (1) ◽

pp. 74 ◽

Cited By ~ 10

Author(s):

Guido Keijzers ◽

Daniela Bakula ◽

Michael Petr ◽

Nils Madsen ◽

Amanuel Teklu ◽

...

Keyword(s):

Cell Cycle ◽

Dna Repair ◽

Dna Replication ◽

Replication Fork ◽

Cellular Transformation ◽

Cell Cycle Checkpoints ◽

Replication Process ◽

Exonuclease 1 ◽

Repair Pathways ◽

Regulatory Functions

Human exonuclease 1 (EXO1), a 5′→3′ exonuclease, contributes to the regulation of the cell cycle checkpoints, replication fork maintenance, and post replicative DNA repair pathways. These processes are required for the resolution of stalled or blocked DNA replication that can lead to replication stress and potential collapse of the replication fork. Failure to restart the DNA replication process can result in double-strand breaks, cell-cycle arrest, cell death, or cellular transformation. In this review, we summarize the involvement of EXO1 in the replication, DNA repair pathways, cell cycle checkpoints, and the link between EXO1 and cancer.

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An Interaction Network of RNA-Binding Proteins Involved in Drosophila Oogenesis

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra119.001912 ◽

2020 ◽

Vol 19 (9) ◽

pp. 1485-1502

Author(s):

Prashali Bansal ◽

Johannes Madlung ◽

Kristina Schaaf ◽

Boris Macek ◽

Fulvia Bono

Keyword(s):

Translational Regulation ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Direct Interaction ◽

Interaction Network ◽

Splicing Factors ◽

Mrna Regulation ◽

Interaction Partners ◽

Maternal Transcripts

During Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.

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167. MUSASHI FAMILY OF RNA BINDING PROTEINS: CELL CYCLE REGULATORS IN SPERMATOGENESIS

Reproduction Fertility and Development ◽

10.1071/srb10abs167 ◽

2010 ◽

Vol 22 (9) ◽

pp. 85

Author(s):

E. A. McLaughlin ◽

B. A. Fraser ◽

V. Pye ◽

M. Bigland ◽

N. A. Siddall ◽

...

Keyword(s):

Cell Cycle ◽

Cell Differentiation ◽

Germ Cell ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Translational Repression ◽

Germ Cell Differentiation ◽

Pachytene Spermatocytes

Mammalian meiosis is a tightly regulated process involving specialized cell cycle progression and morphogenetic changes. We have demonstrated that the Musashi family of RNA binding proteins is implicated in the regulation of spermatogonial stem self renewal and germ cell differentiation. Here we describe the novel mechanism by which the Musashi family proteins, Msi1 and Msi2, act to control exit from spermatogonial mitotic amplification and normal entry into meiosis. Gene and protein analysis indicated overlapping Msi1 and Msi2 profiles in enriched populations of isolated germ cells and reciprocal subcellular expression patterns in spermatogonia and pachytene spermatocytes/ round spermatids in testes sections. Recombinant Msi1 protein-RNA pulldown and microarray analysis coupled with in vitro shRNA knockdown studies in spermatogonial culture and subsequent immunoprecipitation and qPCR established that Msi1 targeted Msi2 mRNA for post transcriptional translational repression. Immunoprecipitation of Msi2 target mRNA and subsequent qPCR together with in vitro shRNA knockdown studies inround spermatidculture identified a cell cycle inhibitor protein CDKN1C (p57kip2) as the principal target of Msi2 translational inhibition. Immunolocalisation of CDKN1C protein indicated that expression of this cell cycle regulator coincided with the nuclear import of Msi1 and the appearance of cytoplasmic Msi2 expression in early pachytene spermatocytes. Using a transgenic Msi1 overexpression mouse model in conjunction with quantitative gene and protein expression, we confirmed Msi1 targeting of Msi2 and subsequent Msi2 targeting of CDKN1C for translational repression in vivo. Ectopic overexpression of Msi1 in germ cellsinduces substantial Msi2 downregulation and aberrant CDKN1C expression, resulting in abnormal spermatogenic differentiation, germ cell apoptosis/arrest and sterility. In conclusion, our results indicate a sophisticated molecular switch encompassing cell cycle protein regulation by Musashi family proteins, is required for normal exit from mitotic division, entry into meiosis and post meiotic germ cell differentiation.

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