scholarly journals Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yarden Katz ◽  
Feifei Li ◽  
Nicole J Lambert ◽  
Ethan S Sokol ◽  
Wai-Leong Tam ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Biology ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
State Regulation ◽  
Ribosome Profiling ◽  
Luminal Cell ◽  
Mesenchymal Transition ◽  
Cell State

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types.

scholarly journals Musashi proteins are post-transcriptional regulators of the epithelial-luminal cell state

2014 ◽  
Author(s):  
Yarden Katz ◽  
Feifei Li ◽  
Nicole Lambert ◽  
Ethan M Sokol ◽  
Wai-Leong Tam ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cell Biology ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Cell Types ◽  
State Regulation ◽  
Luminal Cell ◽  
Mesenchymal Transition ◽  
Cell State

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but mostly absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers, and associated with an epithelial-luminal cell state. Using ribosome footprint profiling and RNA-seq analysis of genetic mouse models in neuronal and mammary cell types, we found that Msis regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT) and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited translation of genes required for EMT, including Jagged1, and repressed EMT in cell culture and in mammary gland in vivo, while knockdown in epithelial cancer cells led to loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program and promote an epithelial-luminal state in both neural and breast cell types.

scholarly journals TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

2020 ◽  
Author(s):  
Hua Jin ◽  
Daxiang Na ◽  
Reazur Rahman ◽  
Weijin Xu ◽  
Allegra Fieldsend ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Growth Control ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Mtor Inhibition ◽  
Mrna Targets ◽  
Specific Mrnas ◽  
Specific Mrna

Abstract4E-BP (eIF4E-BP) represses translation initiation by binding to the 5’cap-binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used hyperTRIBE (Targets of RNA-binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets compared to non-targets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

scholarly journals Characterization of MRP RNA–protein interactions within the perinucleolar compartment

2011 ◽  
Vol 22 (6) ◽  
pp. 858-866 ◽  
Author(s):  
Callie Pollock ◽  
Kelly Daily ◽  
Van Trung Nguyen ◽  
Chen Wang ◽  
Marzena Anna Lewandowska ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Gene Expression Regulation ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Mitochondrial Rna ◽  
Human Telomerase Reverse Transcriptase ◽  
Perinucleolar Compartment

The perinucleolar compartment (PNC) forms in cancer cells and is highly enriched with a subset of polymerase III RNAs and RNA-binding proteins. Here we report that PNC components mitochondrial RNA–processing (MRP) RNA, pyrimidine tract–binding protein (PTB), and CUG-binding protein (CUGBP) interact in vivo, as demonstrated by coimmunoprecipitation and RNA pull-down experiments. Glycerol gradient analyses show that this complex is large and sediments at a different fraction from known MRP RNA–containing complexes, the MRP ribonucleoprotein ribozyme and human telomerase reverse transcriptase. Tethering PNC components to a LacO locus recruits other PNC components, further confirming the in vivo interactions. These interactions are present both in PNC-containing and -lacking cells. High-resolution localization analyses demonstrate that MRP RNA, CUGBP, and PTB colocalize at the PNC as a reticulated network, intertwining with newly synthesized RNA. Furthermore, green fluorescent protein (GFP)–PTB and GFP-CUGBP show a slower rate of fluorescence recovery after photobleaching at the PNC than in the nucleoplasm, illustrating the different molecular interaction of the complexes associated with the PNC. These findings support a working model in which the MRP RNA–protein complex becomes nucleated at the PNC in cancer cells and may play a role in gene expression regulation at the DNA locus that associates with the PNC.

scholarly journals Ribosomes in RNA granules are stalled on mRNA sequences that are consensus sites for FMRP association

2021 ◽  
Author(s):  
Mina N. Anadolu ◽  
Senthilkumar Kailasam ◽  
Konstanze Simbriger ◽  
Jingyu Sun ◽  
Teodora Markova ◽  
...  
Keyword(s):  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Fragile X ◽  
Ribosome Profiling ◽  
Cytoskeletal Proteins ◽  
Rna Granules ◽  
Brain Extracts ◽  
The Brain

AbstractLocal translation in neurons is mediated in part by the reactivation of stalled polysomes. However, the mechanism for stalling of the polysomes is not understood. Stalled polysomes may be enriched within neuronal RNA granules defined by dense collections of compacted ribosomes found in the pellet of sucrose gradients used to separate polysomes from monosomes. We find that this fraction, isolated from P5 rat brains of both sexes, is enriched in proteins implicated in stalled polysome function, such as the fragile X mental retardation protein (FMRP) and Up-frameshift mutation 1 homolog (UPF1). Ribosome profiling of this fraction showed an abundance of footprint reads derived from mRNAs of cytoskeletal proteins implicated in neuronal development and an enrichment of footprint reads on RNA binding proteins. Compared to those usually found in ribosome profiling studies, the footprint reads were more extended on their 3’end and were found in reproducible peaks in the mRNAs. These footprint reads were enriched in motifs previously associated with mRNAs cross-linked to FMRP in vivo, independently linking the ribosomes in the sedimented pellet to the ribosomes associated with FMRP in the cell. The data supports a model in which specific sequences in mRNAs act to stall translation elongation in neurons, attracting FMRP and beginning a process where stalled ribosomes are packaged and transported in RNA granules.Significance StatementThis work finds that neuronal ribosomes in RNA granules are concentrated at consensus sites previously identified through cross-linking FMRP to mRNAs in the brain. This strongly links the compacted ribosomes found in the pellet of sucrose gradients from brain extracts to stalled ribosomes regulated by FMRP and provides important insights into how stalling is accomplished. Many mRNAs important for neurodevelopment are enriched in these ribosomes. These results suggest that many studies on translation in the brain may need to be revised. The larger size of the ribosomal footprints on stalled polysomes and their sedimentation in the pellet of sucrose gradients suggests mRNAs found in these structures have not been assessed in many studies of neuronal translation.

scholarly journals Hsa_circ_0004296 inhibits metastasis of prostate cancer by interacting with EIF4A3 to prevent nuclear export of ETS1 mRNA

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Shiyu Mao ◽  
Wentao Zhang ◽  
Fuhan Yang ◽  
Yadong Guo ◽  
Hong Wang ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Transcriptional Level ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Potential Biomarker ◽  
Gain And Loss

Abstract Background Circular RNAs (circRNAs) have been shown to play vital biological functions in various tumors, including prostate cancer (PCa). However, the roles of circRNAs in the metastasis of PCa remain unclear. In the present study, differentially expressed circRNAs associated with PCa metastasis were screened using high-throughput RNA sequencing, from which hsa_circ_0004296 was identified. Methods Quantitative real-time PCR (qRT-PCR) was used to detect the expression of circ_0004296 in PCa tissues and adjacent normal tissues as well as in blood and urine. Gain and loss of function experiments were performed to investigate the function of circ_0004296 in PCa. Bioinformatics analyses, RNA pull-down assay, and mass spectrometry were conducted to identify RNA-binding proteins. RNA immunoprecipitation and RNA and protein nuclear-cytoplasmic fractionation were performed to investigate the underlying mechanism. A xenograft mouse model was used to analyze the effect of circ_0004296 on PCa growth and metastasis in vivo. Results The expression of circ_0004296 was decreased in PCa tissues, blood, and urine, which was negatively associated with metastasis. Furthermore, gain and loss of function experiments in vitro and in vivo showed that circ_0004296 inhibited the proliferation, migration, invasion, and epithelial-mesenchymal transition of PCa cells. Mechanistically, circ_0004296 regulated host gene ETS1 expression at the post-transcriptional level. EIF4A3 was identified and confirmed as the downstream binding protein of circ_0004296. EIF4A3 expression was significantly upregulated in PCa tissues and associated with PCa metastasis. Silencing EIF4A3 suppressed PCa cell proliferation, migration, invasion, and EMT. Conclusions Circ_0004296 overexpression efficiently inhibited ETS1 mRNA nuclear export by promoting EIF4A3 retention in the nucleus, leading to the downregulation of ETS1 expression and suppression of PCa metastasis; thus, circ_0004296 might be a potential biomarker and therapeutic target for patients with PCa.

scholarly journals TRIBE editing reveals specific mRNA targets of eIF4E-BP in Drosophila and in mammals

2020 ◽  
Vol 6 (33) ◽  
pp. eabb8771 ◽  
Author(s):  
Hua Jin ◽  
Weijin Xu ◽  
Reazur Rahman ◽  
Daxiang Na ◽  
Allegra Fieldsend ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Growth Control ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Mtor Inhibition ◽  
Mrna Targets ◽  
Specific Mrnas ◽  
Specific Mrna

4E-BP (eIF4E-BP) represses translation initiation by binding to the 5′ cap–binding protein eIF4E and inhibiting its activity. Although 4E-BP has been shown to be important in growth control, stress response, cancer, neuronal activity, and mammalian circadian rhythms, it is not understood how it preferentially represses a subset of mRNAs. We successfully used HyperTRIBE (targets of RNA binding proteins identified by editing) to identify in vivo 4E-BP mRNA targets in both Drosophila and mammals under conditions known to activate 4E-BP. The protein associates with specific mRNAs, and ribosome profiling data show that mTOR inhibition changes the translational efficiency of 4E-BP TRIBE targets more substantially compared to nontargets. In both systems, these targets have specific motifs and are enriched in translation-related pathways, which correlate well with the known activity of 4E-BP and suggest that it modulates the binding specificity of eIF4E and contributes to mTOR translational specificity.

scholarly journals Single-cell lineage and transcriptome reconstruction of metastatic cancer reveals selection of aggressive hybrid EMT states

2020 ◽  
Author(s):  
Kamen P Simeonov ◽  
China N Byrns ◽  
Megan L Clark ◽  
Robert J Norgard ◽  
Beth Martin ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Single Cell ◽  
Cancer Cells ◽  
Cell Biology ◽  
Lung Metastases ◽  
Metastatic Cancer ◽  
The Body ◽  
Human Pancreatic Cancer ◽  
Mesenchymal Transition

AbstractMetastatic cancer remains largely incurable due to an incomplete understanding of how cancer cells disseminate throughout the body. However, tools for probing metastatic dissemination and associated molecular changes at high resolution are lacking. Here we present multiplexed, activatable, clonal, and subclonal GESTALT (macsGESTALT), an inducible lineage recorder with concurrent single cell readout of transcriptional and phylogenetic information. By integrating multiple copies of combined static barcodes and evolving CRISPR/Cas9 barcodes, macsGESTALT enables clonal tracing and subclonal phylogenetic reconstruction, respectively. High barcode editing and recovery rates produce deep lineage reconstructions, densely annotated with transcriptomic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we reconstruct dissemination of tens-of-thousands of single cancer cells representing 95 clones and over 6,000 unique subclones across multiple distant sites, e.g. liver and lung metastases. Transcriptionally, cells exist along a continuum of epithelial-to-mesenchymal transition (EMT) in vivo with graded changes in associated signaling, metabolic, and regulatory processes. Lineage analysis reveals that from a majority of non-metastatic, highly epithelial clones, a single dominant clone that has progressed along EMT drives the majority of metastasis. Within this dominant clone a parallel process occurs, where a small number of aggressive subclones drive clonal outgrowth. By precisely mapping subclones along the EMT continuum, we find that size and dissemination gradually increase, peaking at late-hybrid EMT states but precipitously falling once subclones are highly mesenchymal. Late-hybrid EMT states are selected from a predominately epithelial ancestral pool, enabling rapid metastasis but also forcing extensive and continuous population bottlenecking. Notably, late-hybrid gene signatures are associated with decreased survival in human pancreatic cancer, while epithelial, early-hybrid, and highly mesenchymal states are not. Our findings illuminate features of metastasis and EMT with the potential for therapeutic exploitation. Ultimately, macsGESTALT provides a powerful, accessible tool for probing cancer and stem cell biology in vivo.

scholarly journals An alternative splicing switch in FLNB promotes the mesenchymal cell state in human breast cancer

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Ji Li ◽  
Peter S Choi ◽  
Christine L Chaffer ◽  
Katherine Labella ◽  
Justin H Hwang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Alternative Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Human Cancer ◽  
Mesenchymal Cell ◽  
Actin Binding ◽  
Mesenchymal Transition ◽  
Cell State ◽  
A Genome

Alternative splicing of mRNA precursors represents a key gene expression regulatory step and permits the generation of distinct protein products with diverse functions. In a genome-scale expression screen for inducers of the epithelial-to-mesenchymal transition (EMT), we found a striking enrichment of RNA-binding proteins. We validated that QKI and RBFOX1 were necessary and sufficient to induce an intermediate mesenchymal cell state and increased tumorigenicity. Using RNA-seq and eCLIP analysis, we found that QKI and RBFOX1 coordinately regulated the splicing and function of the actin-binding protein FLNB, which plays a causal role in the regulation of EMT. Specifically, the skipping of FLNB exon 30 induced EMT by releasing the FOXC1 transcription factor. Moreover, skipping of FLNB exon 30 is strongly associated with EMT gene signatures in basal-like breast cancer patient samples. These observations identify a specific dysregulation of splicing, which regulates tumor cell plasticity and is frequently observed in human cancer.

scholarly journals Ribonomic Analysis of Human Pum1 Reveals cis-trans Conservation across Species despite Evolution of Diverse mRNA Target Sets

2008 ◽  
Vol 28 (12) ◽  
pp. 4093-4103 ◽  
Author(s):  
Adam R. Morris ◽  
Neelanjan Mukherjee ◽  
Jack D. Keene
Keyword(s):  
Cell Biology ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Target Identification ◽  
Consensus Sequence ◽  
Puf Proteins ◽  
Mrna Targets ◽  
Puf Protein ◽  
Genes Encoding

ABSTRACT PUF family proteins are among the best-characterized regulatory RNA-binding proteins in nonmammalian species, but relatively little is known about mRNA targets or functions of mammalian PUF proteins. In this study, we used ribonomic analysis to identify and analyze mRNAs associated with ribonucleoproteins containing an endogenous human PUF protein, Pum1. Pum1-associated mRNAs were highly enriched for genes encoding proteins that function in transcriptional regulation and cell cycle/proliferation, results consistent with the posttranscriptional RNA regulon model and the proposed ancestral functions of PUF proteins in stem cell biology. Analysis of 3′ untranslated region sequences of Pum1-associated mRNAs revealed a core Pum1 consensus sequence, UGUAHAUA. Pum1 knockdown demonstrated that Pum1 enhances decay of associated mRNAs, and relocalization of Pum1 to stress granules suggested that Pum1 functions in repression of translation. This study is the first in vivo genome-wide mRNA target identification of a mammalian PUF protein and provides direct evidence that human PUF proteins regulate stability of associated mRNAs. Comparison of Pum1-associated mRNAs to mRNA targets of PUF proteins from Saccharomyces cerevisiae and Drosophila melanogaster demonstrates how a well-conserved RNA-binding domain and cognate binding sequence have been evolutionarily rewired to regulate the collective expression of different sets of functionally related genes.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

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