Crucial roles of different RNA-binding hnRNP proteins in Stem Cells

AbstractLIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.

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RALYL increases hepatocellular carcinoma stemness by sustaining the mRNA stability of TGF-β2

Nature Communications ◽

10.1038/s41467-021-21828-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xia Wang ◽

Jin Wang ◽

Yu-Man Tsui ◽

Chaoran Shi ◽

Ying Wang ◽

...

Keyword(s):

Stem Cells ◽

Mrna Stability ◽

Rna Binding ◽

Embryonic Stem ◽

Molecular Characteristics ◽

Specific Gene ◽

Functional Studies ◽

Poor Differentiation ◽

Development In Vitro

AbstractGrowing evidences suggest that cancer stem cells exhibit many molecular characteristics and phenotypes similar to their ancestral progenitor cells. In the present study, human embryonic stem cells are induced to differentiate into hepatocytes along hepatic lineages to mimic liver development in vitro. A liver progenitor specific gene, RALY RNA binding protein like (RALYL), is identified. RALYL expression is associated with poor prognosis, poor differentiation, and metastasis in clinical HCC patients. Functional studies reveal that RALYL could promote HCC tumorigenicity, self-renewal, chemoresistance, and metastasis. Moreover, molecular mechanism studies show that RALYL could upregulate TGF-β2 mRNA stability by decreasing N6-methyladenosine (m6A) modification. TGF-β signaling and the subsequent PI3K/AKT and STAT3 pathways, upregulated by RALYL, contribute to the enhancement of HCC stemness. Collectively, RALYL is a liver progenitor specific gene and regulates HCC stemness by sustaining TGF-β2 mRNA stability. These findings may inspire precise therapeutic strategies for HCC.

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Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells

Cell Reports ◽

10.1016/j.celrep.2021.109198 ◽

2021 ◽

Vol 35 (9) ◽

pp. 109198

Author(s):

Shlomi Dvir ◽

Amir Argoetti ◽

Chen Lesnik ◽

Mark Roytblat ◽

Kohava Shriki ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Embryonic Stem

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Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.1.164-171.1992 ◽

1992 ◽

Vol 12 (1) ◽

pp. 164-171

Author(s):

M J Matunis ◽

W M Michael ◽

G Dreyfuss

Keyword(s):

Hela Cells ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Consensus Sequence ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Predicted Amino Acid Sequence ◽

Cdna Clones ◽

Hnrnp Proteins

At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

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Imp and Syp RNA-binding proteins govern decommissioning ofDrosophilaneural stem cells

Development ◽

10.1242/dev.149500 ◽

2017 ◽

Vol 144 (19) ◽

pp. 3454-3464 ◽

Cited By ~ 31

Author(s):

Ching-Po Yang ◽

Tamsin J. Samuels ◽

Yaling Huang ◽

Lu Yang ◽

David Ish-Horowicz ◽

...

Keyword(s):

Stem Cells ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins

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Function of RNA-binding protein Musashi-1 in stem cells

Experimental Cell Research ◽

10.1016/j.yexcr.2005.02.021 ◽

2005 ◽

Vol 306 (2) ◽

pp. 349-356 ◽

Cited By ~ 243

Author(s):

Hideyuki Okano ◽

Hironori Kawahara ◽

Masako Toriya ◽

Keio Nakao ◽

Shinsuke Shibata ◽

...

Keyword(s):

Stem Cells ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein

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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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daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71

PLoS Genetics ◽

10.1371/journal.pgen.1009881 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1009881

Author(s):

Matthew J. Wirick ◽

Allison R. Cale ◽

Isaac T. Smith ◽

Amelia F. Alessi ◽

Margaret R. Starostik ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Rna Binding ◽

Cell Model ◽

Cell Types ◽

Adult Cell ◽

Heterochronic Gene ◽

Foxo Transcription Factors ◽

Dauer Larvae

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

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daf-16/FOXO blocks adult cell fate in Caenorhabditis elegans dauer larvae via lin-41/TRIM71

10.1101/2021.06.30.450637 ◽

2021 ◽

Author(s):

Matthew J Wirick ◽

Allison R Cale ◽

Isaac T Smith ◽

Amelia F Alessi ◽

Margaret R Starostik ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Rna Binding ◽

Cell Model ◽

Cell Types ◽

Adult Cell ◽

Heterochronic Gene ◽

Foxo Transcription Factors ◽

Dauer Larvae

Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C. elegans, daf-16, promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16/FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p::gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p::gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p::gfp. Adult cell fate and the timing of col-19p::gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29. lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development lin-41 opposes adult cell fate by inhibiting the translation of lin-29, which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p::gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p::gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41, but does not require lin-29. Taken together, this work demonstrates a novel role for daf-16/FOXO as a heterochronic gene that promotes expression of lin-41/TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.

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The U2AF2 /circRNA ARF1/miR-342–3p/ISL2 feedback loop regulates angiogenesis in glioma stem cells

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01691-y ◽

2020 ◽

Vol 39 (1) ◽

Author(s):

Yang Jiang ◽

Jinpeng Zhou ◽

Junshuang Zhao ◽

Haiying Zhang ◽

Long Li ◽

...

Keyword(s):

Stem Cells ◽

Neural Development ◽

Feedback Loop ◽

Rna Binding ◽

Patient Survival ◽

Luciferase Reporter ◽

Circular Rnas ◽

Glioma Stem Cells ◽

Poor Patient ◽

Poor Patient Survival

Abstract Background Glioma is the most common and lethal primary brain tumor in adults, and angiogenesis is one of the key factors contributing to its proliferation, aggressiveness, and malignant transformation. However, the discovery of novel oncogenes and the study of its molecular regulating mechanism based on circular RNAs (circRNAs) may provide a promising treatment target in glioma. Methods Bioinformatics analysis, qPCR, western blotting, and immunohistochemistry were used to detect the expression levels of ISL2, miR-342–3p, circRNA ARF1 (cARF1), U2AF2, and VEGFA. Patient-derived glioma stem cells (GSCs) were established for the molecular experiments. Lentiviral-based infection was used to regulate the expression of these molecules in GSCs. The MTS, EDU, Transwell, and tube formation assays were used to detect the proliferation, invasion, and angiogenesis of human brain microvessel endothelial cells (hBMECs). RNA-binding protein immunoprecipitation, RNA pull-down, dual-luciferase reporter, and chromatin immunoprecipitation assays were used to detect the direct regulation mechanisms among these molecules. Results We first identified a novel transcription factor related to neural development. ISL2 was overexpressed in glioma and correlated with poor patient survival. ISL2 transcriptionally regulated VEGFA expression in GSCs and promoted the proliferation, invasion, and angiogenesis of hBMECs via VEGFA-mediated ERK signaling. Regarding its mechanism of action, cARF1 upregulated ISL2 expression in GSCs via miR-342–3p sponging. Furthermore, U2AF2 bound to and promoted the stability and expression of cARF1, while ISL2 induced the expression of U2AF2, which formed a feedback loop in GSCs. We also showed that both U2AF2 and cARF1 had an oncogenic effect, were overexpressed in glioma, and correlated with poor patient survival. Conclusions Our study identified a novel feedback loop among U2AF2, cARF1, miR-342–3p, and ISL2 in GSCs. This feedback loop promoted glioma angiogenesis, and could provide an effective biomarker for glioma diagnosis and prognostic evaluation, as well as possibly being used for targeted therapy.

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