The RNA-Binding Protein SAM68 regulates cardiomyocyte differentiation by enhancing Gata4 translation

The signal transduction and activation of RNA (STAR) family is composed of RNA-binding proteins (RBPs) that play a central role in mammalian development. Nonetheless, the functions and modes of action that STAR proteins have in lineage specification are still poorly understood. Here, we characterized the role of STAR proteins SAM68 and QUAKING (QKI) in pluripotency and differentiation by performing their depletion through CRISPR-Cas9 in mouse embryonic stem cells (mESCs). Combining RNA-sequencing, ribosome profiling and advanced computational predictions, we found that both SAM68 and QKI regulate the mESCs self-renewal and are indispensable for cardiomyocyte differentiation. At the molecular level, we discovered that SAM68 and QKI antagonistically control the expression of cardiogenic factors. Our calculations indicated that SAM68, unlike QKI, binds the cardiogenic-specific transcription factor Gata4 in a region spanning nucleotides 500 to 1000 of the mRNA corresponding to part of the 5' untranslated region and the first exon. We validated the predictions by electrophoretic mobility shift assay and RNA immunoprecipitation showing that SAM68 controls the translation of Gata4 during mESCs differentiation towards the cardiomyocyte lineage.

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Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells

10.1101/560383 ◽

2019 ◽

Author(s):

Samudyata ◽

Paulo P. Amaral ◽

Pär G. Engström ◽

Samuel C. Robson ◽

Michael L. Nielsen ◽

...

Keyword(s):

Transcriptional Repression ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Messenger Rnas ◽

Heterochromatin Protein 1 ◽

Non Coding Rna ◽

Rna Immunoprecipitation ◽

Mes Cells

AbstractSox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role of Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interact with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but might rather regulate other processes in the nucleoplasm.Summary blurbSox2 interacts with RNA-binding proteins and diverse RNAs

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Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

Scientific Reports ◽

10.1038/s41598-021-87097-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yogeeshwar Ajjugal ◽

Narendar Kolimi ◽

Thenmalarchelvi Rathinavelan

Keyword(s):

Rna Binding ◽

Trinucleotide Repeat ◽

Rna Binding Proteins ◽

Fragile X ◽

Repeat Expansion ◽

Mobility Shift ◽

Cgg Repeat ◽

Dna And Rna ◽

Structural Choice ◽

Sense Strand

AbstractCGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

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RNA recognition and self-association of CPEB4 is mediated by its tandem RRM domains

Nucleic Acids Research ◽

10.1093/nar/gku700 ◽

2014 ◽

Vol 42 (15) ◽

pp. 10185-10195 ◽

Cited By ~ 4

Author(s):

Constanze Schelhorn ◽

James M.B. Gordon ◽

Lidia Ruiz ◽

Javier Alguacil ◽

Enrique Pedroso ◽

...

Keyword(s):

Rna Binding ◽

Titration Calorimetry ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Mobility Shift ◽

Cytoplasmic Polyadenylation ◽

C Terminus ◽

Self Association ◽

Mobility Shift Assay ◽

A Minor

Abstract Cytoplasmic polyadenylation is regulated by the interaction of the cytoplasmic polyadenylation element binding proteins (CPEB) with cytoplasmic polyadenylation element (CPE) containing mRNAs. The CPEB family comprises four paralogs, CPEB1–4, each composed of a variable N-terminal region, two RNA recognition motif (RRM) and a C-terminal ZZ-domain. We have characterized the RRM domains of CPEB4 and their binding properties using a combination of biochemical, biophysical and NMR techniques. Isothermal titration calorimetry, NMR and electrophoretic mobility shift assay experiments demonstrate that both the RRM domains are required for an optimal CPE interaction and the presence of either one or two adenosines in the two most commonly used consensus CPE motifs has little effect on the affinity of the interaction. Both the single RRM1 and the tandem RRM1–RRM2 have the ability to dimerize, although representing a minor population. Self-association does not affect the proteins’ ability to interact with RNA as demonstrated by ion mobility–mass spectrometry. Chemical shift effects measured by NMR of the apo forms of the RRM1–RRM2 samples indicate that the two domains are orientated toward each other. NMR titration experiments show that residues on the β-sheet surface on RRM1 and at the C-terminus of RRM2 are affected upon RNA binding. We propose a model of the CPEB4 RRM1–RRM2–CPE complex that illustrates the experimental data.

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FUS driven circCNOT6L biogenesis in mouse and human spermatozoa supports zygote development

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-04054-8 ◽

2021 ◽

Author(s):

Teresa Chioccarelli ◽

Geppino Falco ◽

Donato Cappetta ◽

Antonella De Angelis ◽

Luca Roberto ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Rna Binding ◽

Cannabinoid Receptor ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Circular Rna ◽

Physical Interaction ◽

Knock Out ◽

Maternal Contribution ◽

Zygote Development

AbstractCircular RNA (circRNA) biogenesis requires a backsplicing reaction, promoted by inverted repeats in cis-flanking sequences and trans factors, such as RNA-binding proteins (RBPs). Among these, FUS plays a key role. During spermatogenesis and sperm maturation along the epididymis such a molecular mechanism has been poorly explored. With this in mind, we chose circCNOT6L as a study case and wild-type (WT) as well as cannabinoid receptor type-1 knock-out (Cb1−/−) male mice as animal models to analyze backsplicing mechanisms. Our results suggest that spermatozoa (SPZ) have an endogenous skill to circularize mRNAs, choosing FUS as modulator of backsplicing and under CB1 stimulation. A physical interaction between FUS and CNOT6L as well as a cooperation among FUS, RNA Polymerase II (RNApol2) and Quaking (QKI) take place in SPZ. Finally, to gain insight into FUS involvement in circCNOT6L biogenesis, FUS expression was reduced through RNA interference approach. Paternal transmission of FUS and CNOT6L to oocytes during fertilization was then assessed by using murine unfertilized oocytes (NF), one-cell zygotes (F) and murine oocytes undergoing parthenogenetic activation (PA) to exclude a maternal contribution. The role of circCNOT6L as an active regulator of zygote transition toward the 2-cell-like state was suggested using the Embryonic Stem Cell (ESC) system. Intriguingly, human SPZ exactly mirror murine SPZ.

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Systematic discovery of endogenous human ribonucleoprotein complexes

10.1101/480061 ◽

2018 ◽

Cited By ~ 2

Author(s):

Anna L. Mallam ◽

Wisath Sae-Lee ◽

Jeffrey M. Schaub ◽

Fan Tu ◽

Anna Battenhouse ◽

...

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Protein Complexes ◽

Embryonic Stem ◽

Human Protein ◽

Disease States ◽

Ribonucleoprotein Complexes ◽

Associated Proteins ◽

Domains Of Life ◽

Factor C

AbstractRNA-binding proteins (RBPs) play essential roles in biology and are frequently associated with human disease. While recent studies have systematically identified individual RBPs, their higher order assembly intoRibonucleoprotein (RNP) complexes has not been systematically investigated. Here, we describe a proteomics method for systematic identification of RNP complexes in human cells. We identify 1,428 protein complexes that associate with RNA, indicating that over 20% of known human protein complexes contain RNA. To explore the role of RNA in the assembly of each complex, we identify complexes that dissociate, change composition, or form stable protein-only complexes in the absence of RNA. Importantly, these data also provide specific novel insights into the function of well-studied protein complexes not previously known to associate with RNA, including replication factor C (RFC) and cytokinetic centralspindlin complex. Finally, we use our method to systematically identify cell-type specific RNA-associated proteins in mouse embryonic stem cells. We distribute these data as a resource, rna.MAP (rna.proteincomplexes.org) which provides a comprehensive dataset for the study of RNA-associated protein complexes. Our system thus provides a novel methodology for further explorations across human tissues and disease states, as well as throughout all domains of life.SummaryAn exploration of human protein complexes in the presence and absence of RNA reveals endogenous ribonucleoprotein complexes

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

eLife ◽

10.7554/elife.03915 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 55

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.

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Function of Pumilio Genes in Human Embryonic Stem Cells and Their Effect in Stemness and Cardiomyogenesis

10.1101/751537 ◽

2019 ◽

Author(s):

Isabelle Leticia Zaboroski Silva ◽

Anny Waloski Robert ◽

Guillermo Cabrera Cabo ◽

Lucia Spangenberg ◽

Marco Augusto Stimamiglio ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Mrna Levels ◽

Human Escs ◽

Mrna Targets ◽

Cardiomyogenic Differentiation

AbstractPosttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA binding proteins (RBPs) that orchestrate the expression of these molecules. A family of RBPs, known as PUF (Pumilio-FBF), is highly conserved among species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first demonstrated the influence of the silencing of PUM1 and PUM2 on pluripotency genes. OCT4 and NANOG mRNA levels decreased significantly with the knockdown of Pumilio, suggesting that PUMILIO proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that the hESCs silenced for PUM1 and 2 exhibited an improvement in efficiency of in vitro cardiomyogenic differentiation. Using in silico analysis, we identified mRNA targets of PUM1 and PUM2 expressed during cardiomyogenesis. With the reduction of PUM1 and 2, these target mRNAs would be active and could be involved in the progression of cardiomyogenesis.

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

10.1101/2020.02.24.962852 ◽

2020 ◽

Cited By ~ 2

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.

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Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

Molecular and Cellular Biology ◽

10.1128/mcb.24.14.6241-6252.2004 ◽

2004 ◽

Vol 24 (14) ◽

pp. 6241-6252 ◽

Cited By ~ 106

Author(s):

Kristina L. Carroll ◽

Dennis A. Pradhan ◽

Josh A. Granek ◽

Neil D. Clarke ◽

Jeffry L. Corden

Keyword(s):

Rna Polymerase Ii ◽

Binding Sites ◽

Rna Binding ◽

Rna Binding Proteins ◽

Large Degree ◽

Sequence Motifs ◽

Mobility Shift ◽

Nascent Transcript ◽

Pol Ii ◽

Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

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RNA Binding Proteins Control Transdifferentiation of Hepatic Stellate Cells into Myofibroblasts

Cellular Physiology and Biochemistry ◽

10.1159/000491987 ◽

2018 ◽

Vol 48 (3) ◽

pp. 1215-1229 ◽

Cited By ~ 5

Author(s):

Sihyung Wang ◽

Youngmi Jung ◽

Jeongeun Hyun ◽

Matthew Friedersdorf ◽

Seh-Hoon Oh ◽

...

Keyword(s):

Hepatic Stellate Cells ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Stellate Cells ◽

Fibrous Matrix ◽

Rna Immunoprecipitation ◽

Physical Interactions ◽

Β Receptor ◽

Dependent Mechanism

Background/Aims: Myofibroblasts (MF) derived from quiescent nonfibrogenic hepatic stellate cells (HSC) are the major sources of fibrous matrix in cirrhosis. Because many factors interact to regulate expansion and regression of MF-HSC populations, efforts to prevent cirrhosis by targeting any one factor have had limited success, motivating research to identify mechanisms that integrate these diverse inputs. As key components of RNA regulons, RNA binding proteins (RBPs) may fulfill this function by orchestrating changes in the expression of multiple genes that must be coordinately regulated to affect the complex phenotypic modifications required for HSC transdifferentiation. Methods: We profiled the transcriptomes of quiescent and MF-HSC to identify RBPs that were differentially-expressed during HSC transdifferentiation, manipulated the expression of the most significantly induced RBP, insulin like growth factor 2 binding protein 3 (Igf2bp3), and evaluated transcriptomic and phenotypic effects. Results: Depleting Igf2bp3 changed the expression of thousands of HSC genes, including multiple targets of TGF-β signaling, and caused HSCs to reacquire a less proliferative, less myofibroblastic phenotype. RNA immunoprecipitation assays demonstrated that some of these effects were mediated by direct physical interactions between Igf2bp3 and mRNAs that control proliferative activity and mesenchymal traits. Inhibiting TGF-β receptor-1 signaling revealed a microRNA-dependent mechanism that induces Igf2bp3. Conclusions: The aggregate results indicate that HSC transdifferentiation is ultimately dictated by Igf2bp3-dependent RNA regulons and thus, can be controlled simply by manipulating Igf2bp3.

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