Abstract 189: HuR, RNA Stabilizing Protein, Regulation of Pluripotency and Differentiation in iPSCs

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Sahana Suresh Babu ◽  
Johnson Rajasingh ◽  
Wing Tak Wong ◽  
Prasanna Krishnamurthy
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Dermal Fibroblast ◽  
Critical Role ◽  
Human Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
General Observation ◽  
Transcript Stability ◽  
Differentiated Cells ◽  
Induced Pluripotent

Background: The Hu family of RNA-binding proteins, HuR (also known as ELAVL1 or human embryonic lethal abnormal vision-like protein), binds to the 3’-untranslated region of mRNAs and regulates transcript stability and translation. Global deletion of HuR is embryonically lethal in mice and plays a critical role in progenitor cell survival and biology. Induced-pluripotent stem cells (iPSC) have distinct transcriptional machinery for the maintenance of pluripotency and achievement of differentiation. However, the exact role of HuR in pluripotency or differentiation of iPSC to cardiomyocytes (iCM) remains unclear. Methods: HuR knockdown in human dermal fibroblast-derived iPSCs was achieved by CRISPR/Cas9 or lentiviral shRNA transduction and subsequently differentiated into cardiomyocytes (iCM). Then, the expression of HuR, pluripotency and cardiomyocyte markers were evaluated on days 0, 1, 3, 6, 8 and 17 following the initiation of differentiation. Results: At basal level, HuR expression was higher in the iPSCs compared to dermal fibroblasts. Upon differentiation of iPSCs into iCM, HuR mRNA expression gradually reduced with significantly lower levels on day 17. As expected, pluripotency markers gradually reduced upon differentiation with significantly lower levels from day 6 onwards. We observed a corresponding increase in ISL1, MESP1 (mesoderm/cardiac progenitor markers) from day 3 through day 8 with a steep fall from day 8 to day 17. This was associated with Myosin light chain-2V and GATA4 expression increases from day 8 through day 17. Interestingly, knockdown of HuR resulted in clumps of colonies with differentiated cells and a corresponding increase in cardiac-troponin positive cells. However, as a general observation, HuR knockdown reduced beating intensity compared to wild type cells. Conclusions: Based on these data, we could speculate that HuR might be necessary for maintenance of pluripotency and loss of which renders cells to differentiate in culture. HuR knockdown yields higher number of c-troponin positive cells but its effect on functional maturity of iCM needs to be further evaluated.

scholarly journals RNA-binding proteins La and HuR cooperatively modulate translation repression of PDCD4 mRNA

2020 ◽  
pp. jbc.RA120.014894
Author(s):  
Ravi Kumar ◽  
Dipak Kumar Poria ◽  
Partho Sarothi Ray
Keyword(s):  
Inflammatory Response ◽  
Chronic Inflammation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Regulation Of Gene Expression ◽  
Mrna Translation ◽  
Suppressor Gene ◽  
Cooperative Action ◽  
Translation Repression

Post-transcriptional regulation of gene expression plays a critical role in controlling the inflammatory response. An uncontrolled inflammatory response results in chronic inflammation, often leading to tumorigenesis. Programmed cell death 4 (PDCD4) is a pro-inflammatory tumor-suppressor gene which helps to prevent the transition from chronic inflammation to cancer. PDCD4 mRNA translation is regulated by an interplay between the oncogenic microRNA miR-21 and the RNA-binding protein (RBP) HuR in response to LPS stimulation, but the role of other regulatory factors remain unknown. Here we report that the RBP Lupus antigen (La) interacts with the 3’UTR of PDCD4 mRNA and prevents miR-21-mediated translation repression. While LPS causes nuclear-cytoplasmic translocation of HuR, it enhances cellular La expression. Remarkably, La and HuR were found to bind cooperatively to the PDCD4 mRNA and mitigate miR-21-mediated translation repression. The cooperative action of La and HuR reduced cell proliferation and enhanced apoptosis, reversing the pro-oncogenic function of miR-21. Together, these observations demonstrate a cooperative interplay between two RBPs, triggered differentially by the same stimulus, which exerts a synergistic effect on PDCD4 expression and thereby helps maintain a balance between inflammation and tumorigenesis.

scholarly journals Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells

2021 ◽  
Vol 43 (3) ◽  
pp. 2124-2134
Author(s):  
Hyun Soo Lee ◽  
Jeewon Mok ◽  
Choun-Ki Joo
Keyword(s):  
Progenitor Cells ◽  
Dermal Fibroblast ◽  
Ips Cells ◽  
Human Dermal Fibroblast ◽  
Corneal Epithelial ◽  
Specific Medium ◽  
Morphogenetic Protein ◽  
Human Ips Cells ◽  
Limbal Stem Cell ◽  
Induced Pluripotent

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

A Potential Therapeutic Target RNA-binding Protein, Arid5a for the Treatment of Inflammatory Disease Associated with Aberrant Cytokine Expression

2018 ◽  
Vol 24 (16) ◽  
pp. 1766-1771 ◽  
Author(s):  
Kazuya Masuda ◽  
Tadamitsu Kishimoto
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Severe Sepsis ◽  
Inflammatory Cytokines ◽  
Binding Proteins ◽  
Rna Binding ◽  
Cytokine Production ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Transcriptional Level

Background: Infection, tissue damage and aging can cause inflammation with high levels of inflammatory cytokines. Overproduction of inflammatory cytokines often leads to systemic inflammatory response syndrome (SIRS), severe sepsis, and septic shock. However, prominent therapeutic targets have not been found, although the incidence of sepsis is likely to increase annually. Our recent studies indicate that some RNA-binding proteins, which control gene expression of inflammatory cytokines at the post-transcriptional level, may play a critical role in inflammatory diseases such as sepsis. Results: 1) One of the RNA-binding proteins, AT-rich interactive domain-containing 5a (Arid5a) promotes cytokine production through control of mRNA half-lives of pro-inflammatory molecules such as IL-6, STAT3, T-bet, and OX40 in activated macrophages and T cells. Arid5a KO mice are refractory to endotoxin shock, bleomycininduced lung injury, and inflammatory autoimmune disease. 2) Chlorpromazine (CPZ), which is recognized as a psychotic drug, impairs post-transcriptional gene expression of Il6 in LPS-stimulated macrophages: CPZ inhibits the binding activity of Arid5a to the 3’UTR of Il6 mRNA, thereby destabilizing Il6 mRNA possibly through suppression of Arid5a expression. 3) CPZ has strong suppressive effects on cytokine production such as TNF-α in vivo. Mice with treatment of CPZ are resistant to lipopolysaccharide (LPS)-induced shock. Conclusion: Thus, Arid5a contributes to the activation of macrophages and T cells through positive control of mRNA half-lives of inflammatory cytokines and its related molecules, which might lead to cytokine storm. Interestingly, Arid5a was identified from an inhibitory effect of CPZ on IL-6 production in macrophages activated by LPS. Therefore, CPZ derivatives or Arid5a inhibitors may have a potential to suppress severe sepsis through control of post-transcriptional gene expression.

scholarly journals A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

2020 ◽  
Vol 295 (42) ◽  
pp. 14291-14304
Author(s):  
Kathrin Bajak ◽  
Kevin Leiss ◽  
Christine Clayton ◽  
Esteban Erben
Keyword(s):  
Gene Expression ◽  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Affinity Purification ◽  
Critical Role ◽  
Rna Binding Protein ◽  
Mrna Translation

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5′-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of “halfmer” disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

scholarly journals Wound Healing Potential of Spirulina Protein on CCD-986sk Cells

Marine Drugs ◽  
2019 ◽  
Vol 17 (2) ◽  
pp. 130
Author(s):  
Ping Liu ◽  
Jeong-Wook Choi ◽  
Min-Kyeong Lee ◽  
Youn-Hee Choi ◽  
Taek-Jeong Nam
Keyword(s):  
Wound Healing ◽  
Dermal Fibroblast ◽  
Fibroblast Cell ◽  
Underlying Mechanism ◽  
Human Dermal Fibroblast ◽  
Dermal Fibroblasts ◽  
Cyclin Dependent Kinase ◽  
Pi3k Inhibitor Ly294002 ◽  
And Migration ◽  
Proliferation And Migration

Wound healing is a dynamic and complex process. The proliferation and migration of dermal fibroblasts are crucial for wound healing. Recent studies have indicated that the extracts from Spirulina platensis have a positive potential for wound healing. However, its underlying mechanism is not fully understood. Our previous study showed that spirulina crude protein (SPCP) promoted the viability of human dermal fibroblast cell line (CCD-986sk cells). In this study, we further investigated the wound healing effect and corresponding mechanisms of SPCP on CCD-986sk cells. Bromodeoxyuridine (BrdU) assay showed that SPCP promoted the proliferation of CCD-986sk cells. The wound healing assay showed that SPCP promoted the migration of CCD-986sk cells. Furthermore, cell cycle analysis demonstrated that SPCP promoted CCD-986sk cells to enter S and G2/M phases from G0/G1 phase. Western blot results showed that SPCP significantly upregulated the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (Cdk2), cyclin-dependent kinase 4 (Cdk4), and cyclin-dependent kinase 6 (Cdk6), as well as inhibited the expression of CDK inhibitors p21 and p27 in CCD-986sk cells. In the meanwhile, SPCP promoted the phosphorylation and activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt). However, the phosphorylation of Akt was significantly blocked by PI3K inhibitor (LY294002), which in turn reduced the SPCP-induced proliferation and migration of CCD-986sk cells. Therefore, the results presenting in this study suggested that SPCP can promote the proliferation and migration of CCD-986sk cells; the PI3K/Akt signaling pathway play a positive and important role in these processes.

scholarly journals Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene

2020 ◽  
Vol 21 (12) ◽  
pp. 4302 ◽  
Author(s):  
Debojyoti Das ◽  
Aniruddha Das ◽  
Mousumi Sahu ◽  
Smruti Sambhav Mishra ◽  
Shaheerah Khan ◽  
...  
Keyword(s):  
Rna Binding ◽  
Cell Function ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Large Family ◽  
Normal Diet ◽  
Circular Rnas ◽  
Sequencing Data ◽  
Reverse Transcription Pcr ◽  
Β Cell Function

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic β-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 (Ins2) gene and are termed as circular intronic (ci)-Ins2. The expression of ci-Ins2 transcripts in mouse pancreatic islets, and βTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of ci-Ins2 was altered in βTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with ci-Ins2 and their flanking region, suggesting their role in the ci-Ins2 function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with ci-Ins2. Gene ontology and pathway analysis of genes targeted by miRNAs associated with ci-Ins2 suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially ci-Ins2 transcripts, may regulate β-cell function and may play a critical role in the development of diabetes.

scholarly journals RBPMetaDB: A comprehensive annotation of mouse RNA-Seq datasets with perturbations of RNA-binding proteins

2018 ◽  
Author(s):  
Jin Li ◽  
Su-Ping Deng ◽  
Jacob Vieira ◽  
James Thomas ◽  
Valerio Costa ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Dna Binding ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Experimental Studies ◽  
Easy Access ◽  
Rna Seq ◽  
Mrna Stabilization

AbstractRNA-binding proteins may play a critical role in gene regulation in various diseases or biological processes by controlling post-transcriptional events such as polyadenylation, splicing, and mRNA stabilization via binding activities to RNA molecules. Due to the importance of RNA-binding proteins in gene regulation, a great number of studies have been conducted, resulting in a large amount of RNA-Seq datasets. However, these datasets usually do not have structured organization of metadata, which limits their potentially wide use. To bridge this gap, the metadata of a comprehensive set of publicly available mouse RNA-Seq datasets with perturbed RNA-binding proteins were collected and integrated into a database called RBPMetaDB. This database contains 278 mouse RNA-Seq datasets for a comprehensive list of 163 RNA-binding proteins. These RNA-binding proteins account for only ∼10% of all known RNA-binding proteins annotated in Gene Ontology, indicating that most are still unexplored using high-throughput sequencing. This negative information provides a great pool of candidate RNA-binding proteins for biologists to conduct future experimental studies. In addition, we found that DNA-binding activities are significantly enriched among RNA-binding proteins in RBPMetaDB, suggesting that prior studies of these DNA- and RNA-binding factors focus more on DNA-binding activities instead of RNA-binding activities. This result reveals the opportunity to efficiently reuse these data for investigation of the roles of their RNA-binding activities. A web application has also been implemented to enable easy access and wide use of RBPMetaDB. It is expected that RBPMetaDB will be a great resource for improving understanding of the biological roles of RNA-binding proteins.Database URL: http://rbpmetadb.yubiolab.org

scholarly journals Dermal Olfactory Receptor OR51B5 Is Essential for Survival and Collagen Synthesis in Human Dermal Fibroblast (Hs68 Cells)

2021 ◽  
Vol 22 (17) ◽  
pp. 9273
Author(s):  
Bomin Son ◽  
Wesuk Kang ◽  
Soyoon Park ◽  
Dabin Choi ◽  
Taesun Park
Keyword(s):  
Cell Survival ◽  
Collagen Synthesis ◽  
Dermal Fibroblast ◽  
Olfactory Receptors ◽  
Camp Response Element Binding ◽  
Human Dermal Fibroblast ◽  
Skin Aging ◽  
Dermal Fibroblasts ◽  
Nasal Tissue ◽  
Extracellular Matrix Components

Skin dermis comprises extracellular matrix components, mainly collagen fibers. A decrease in collagen synthesis caused by several factors, including ultraviolet (UV) irradiation and stress, eventually causes extrinsic skin aging. Olfactory receptors (ORs) were initially considered to be specifically expressed in nasal tissue, but several ORs have been reported to be present in other tissues, and their biological roles have recently received increasing attention. In this study, we aimed to characterize the role of ORs in cell survival and collagen synthesis in dermal fibroblasts. We confirmed that UVB irradiation and dexamethasone exposure significantly decreased cell survival and collagen synthesis in Hs68 dermal fibroblasts. Moreover, we demonstrated that the mRNA expression of 10 ORs detectable in Hs68 cells was significantly downregulated in aged conditions compared with that in normal conditions. Thereafter, by individual knockdown of the 10 candidate ORs, we identified that only OR51B5 knockdown leads to a reduction of cell survival and collagen synthesis. OR51B5 knockdown decreased cAMP levels and dampened the downstream protein kinase A/cAMP-response element binding protein pathway, downregulating the survival- and collagen synthesis-related genes in the dermal fibroblasts. Therefore, OR51B5 may be an interesting candidate that plays a role in cell survival and collagen synthesis.

scholarly journals Effect of MMP/TIMP Balancing of Cynoglossus semilaevis Shell Extracts on Skin Protection

Fishes ◽  
2021 ◽  
Vol 6 (3) ◽  
pp. 34
Author(s):  
Soo-Cheol Choi ◽  
In-Ah Lee
Keyword(s):  
Skin Diseases ◽  
Dermal Fibroblast ◽  
Functional Materials ◽  
Human Dermal Fibroblast ◽  
Cynoglossus Semilaevis ◽  
Dermal Fibroblasts ◽  
Skin Tissue ◽  
Dependent Manner ◽  
Functional Material ◽  
Concentration Dependent Manner

Cynoglossus semilaevis shell is a by-product of the Cynoglossus semilaevis, a species of fish mainly distributed along the west coast of Korea. As its skin is very tough and difficult to process, it is not useful as food. For this reason, most of it is discarded except for a small amount that is used as feed, which results in environmental pollution. Considering this, there is a need for research on the development of functional materials using Cynoglossus semilaevis shell. This study focused on the mechanism of in vitro expression function of Cynoglossus semilaevis shell extract (CSE) for skin tissue in human dermal fibroblasts that induced or did not induce wrinkles by UV-B irradiation and aims to use it as a functional material for human skin beauty or wrinkle improvement through extraction and purification. According to the ELISA results using human dermal fibroblast cells, CSE reduced MMP-1 and elastase activity by up to 21.89% and 12.04%, respectively, in a concentration-dependent manner, and increased PIP synthesis by up to 62.24% in a concentration-dependent manner. The RT-PCR test results using mRNA showed the MMP-1, 2, and 3 expression levels were suppressed in the CSE-treated group compared to the UVB-induced group and caused a concentration-dependent increase in TIMP-1 in the CSE-treat group. These results suggest that CSE can maintain and improve skin tissue conditions through MMP/TIMP balancing in human dermal fibroblast cell lines and indicate its potential as a functional material for improving skin diseases and suppressing photo-aging.

scholarly journals Ectopic expression ofDrosophilaELAV and human HuD inDrosophilawing disc cells reveals functional distinctions and similarities

2002 ◽  
Vol 115 (11) ◽  
pp. 2413-2421 ◽  
Author(s):  
Gakuta Toba ◽  
Jan Qui ◽  
Sandhya P. Koushika ◽  
Kalpana White
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Rna Binding Proteins ◽  
Mrna Level ◽  
Ectopic Expression ◽  
Wing Disc ◽  
Transcript Stability ◽  
Endogenous Expression ◽  
Disc Cells

Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements(AREs) in their 3′ untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes,which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame(ORF); (2) with a constitutively spliced intron inserted into the ORF; and (3)with the intron nASI whose splicing is regulated in neurons by ELAV,inserted into the ORF. The two sets differed from each other only in their 3′UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. Our results show that:(1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3′UTR, but not of transgenes with Act5C 3′UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm,ELAV is localized to both the cytoplasm and the nucleus.

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