scholarly journals The RNA-binding protein Puf5 buffers mRNA levels against chromatin-mediated changes in nascent transcription

2020 ◽  
Author(s):  
David Z. Kochan ◽  
Julia S. P. Mawer ◽  
Kiril Tishinov ◽  
Swati Parekh ◽  
Jennifer Massen ◽  
...  
Keyword(s):  
Rna Binding ◽  
Genetic Interaction ◽  
Synthetic Lethality ◽  
Mrna Export ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Ribosomal Protein Genes ◽  
Feedback Mechanisms ◽  
Mrna Targets ◽  
Widespread Phenomenon

ABSTRACTGene expression is a dynamic process regulated at all stages, starting with opening of chromatin, transcription, and continuing with mRNA export, translation and, finally, degradation. While there are feedback mechanisms within the system, it is not clear whether these extend to crosstalk between chromatin architecture and mRNA decay. Here, we show that changes in nascent transcription, mediated by mutating H3K56 to alanine, are post-transcriptionally buffered by the Pumilio protein Puf5, which stabilizes transcripts in a context-dependent manner. Depleting Puf5 in an H3K56A background leads to synthetic lethality. This genetic interaction can be explained by a decrease in translation due to downregulation of its direct mRNA targets, largely consisting of ribosomal protein genes. Importantly, we show that this post-transcriptional buffering is not only linked to H3K56A, but may be a more widespread phenomenon that also buffers against an increase in nascent RNA transcription in order to maintain physiological mRNA levels and cellular homeostasis.

scholarly journals The RNA-binding protein Puf5 contributes to buffering of mRNA upon chromatin-mediated changes in nascent transcription

2021 ◽  
Author(s):  
David Z. Kochan ◽  
Julia S. P. Mawer ◽  
Jennifer Massen ◽  
Kiril Tishinov ◽  
Swati Parekh ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Rna Binding ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Feedback Mechanisms ◽  
Mrna Targets ◽  
Genome Wide ◽  
A Genome ◽  
Mrna Surveillance ◽  
Global Increase

Gene expression involves regulation of chromatin structure and transcription, as well as processing of the transcribed mRNA. While there are feedback mechanisms, it is not clear whether these include crosstalk between chromatin architecture and mRNA decay. To address this, we performed a genome-wide genetic screen using a yeast strain harbouring the H3K56A mutation known to perturb chromatin structure and nascent transcription. We identified Puf5 as essential in an H3K56A background. Depletion of Puf5 in this background leads to downregulation of Puf5 targets. We suggest that Puf5 plays a role in post-transcriptional buffering of mRNAs and support this by transcriptional shutoff experiments in which Puf5 mRNA targets are degraded slower in H3K56A compared to wildtype. Finally, we show that post-transcriptional buffering of Puf5 targets is widespread and does not occur only in an H3K56A mutant, but also in an H3K4R background, which leads to a global increase in nascent transcription. Our data suggest that Puf5 determines the fate of its mRNA targets in a context-dependent manner acting as an mRNA surveillance hub balancing de-regulated nascent transcription to maintain physiological mRNA levels.

scholarly journals Mex67p of Schizosaccharomyces pombeInteracts with Rae1p in Mediating mRNA Export

2000 ◽  
Vol 20 (23) ◽  
pp. 8767-8782 ◽  
Author(s):  
Jin Ho Yoon ◽  
Dona C. Love ◽  
Anjan Guhathakurta ◽  
John A. Hanover ◽  
Ravi Dhar
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Synthetic Lethality ◽  
Sequence Similarity ◽  
Mrna Export ◽  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Multicopy Suppressor ◽  
Accessory Factor

ABSTRACT We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 tsmutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast toscMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with therae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of therae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

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

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.

Abstract 19285: Notch1 Haploinsufficiency Increases Risk of Congenital Heart Defects in the Setting of Maternal Diabetes by an Epigenetic Mechanism

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Madhumita Basu ◽  
Kevin Bosse ◽  
Vidu Garg
Keyword(s):  
Signaling Pathway ◽  
Congenital Heart ◽  
Genetic Interaction ◽  
Heart Defects ◽  
Maternal Diabetes ◽  
Epigenetic Mechanism ◽  
Luciferase Reporter ◽  
Environment Interaction ◽  
Mrna Levels ◽  
Dependent Manner

Congenital heart disease (CHD) is the most common type of birth defect. Epidemiologic studies have shown the importance of genetic and environmental factors in the multifactorial etiology of CHD. Maternal diabetes mellitus (DM) is one of the non-genetic risk factors that predisposes to CHD predominantly cardiac septation and cardiac outflow tract defects. DM is known to be associated with endothelial cell dysfunction and we recently demonstrated a genetic interaction between endothelial nitric oxide synthase and Notch1, which encodes a receptor that functions in an important cardiac developmental signaling pathway. We hypothesized that maternal DM in the setting of Notch1 heterozygosity of the developing embryo will predispose to CHD. Notch1 +/- embryos (E13.5) exposed to maternal DM demonstrated an increased incidence (86%) of ventricular septal defects compared to wildtype littermates (22%) (Table). Gene expression studies in non-diabetic wildtype, diabetic wildtype and Notch1 +/- embryos showed DM was associated with decreased Notch1 mRNA levels and upregulation in Jarid2, a histone H3K9 demethylase known to regulate Notch1. In H9C2 and endocardial-derived cells and chick embryos, we showed that hyperglycemia led to decreased expression of Notch1 and its downstream targets in a dose-dependent manner. Similarly, Jarid2 mRNA levels increased with high glucose. Furthermore, we found reduced luciferase reporter activity in cells transfected with a constitutively active Notch1 intracellular domain with hyperglycemia. Studies demonstrating the relative enrichment of Jarid2 on Notch1 locus with hyperglycemia by ChIP-qPCR will also be presented. Our findings reveal that maternal DM disrupts cardiac development by deregulating the Notch1 signaling pathway and suggest that this gene-environment interaction is mediated by an epigenetic mechanism involving Jarid2 providing the first mechanistic insights for this association.

scholarly journals RNA-Binding Protein Musashi-2 Inhibits Aldosterone Production

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A815-A815
Author(s):  
Kathryn Bartholomay ◽  
Amber Baldwin ◽  
Neelanjan Mukherjee
Keyword(s):  
Molecular Mechanism ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Steroid Hormone ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Hormone Synthesis ◽  
Aldosterone Production

Abstract The adrenal cortex is the site of steroid hormone synthesis. These hormones control important physiological processes like metabolism, blood pressure and volume, and sexual characteristic development. While the signaling pathways, transcription factors, and steroidogenic enzymes are well-characterized, surprisingly little is known about the contribution RNA-binding proteins (RBPs). RBPs exert post-transcriptional control by interacting with specific elements within target mRNAs. Here we focus on the RBP, Mushashi-2 (MSI2), which binds to UAG sequences in the 3’UTR of its target transcripts. MSI2 is required for development of steroidogenic tissues which is consistent with its higher mRNA levels in human ovaries and testis. MSI2 also exhibits high expression levels in human adrenal tissue and the immortalized human adrenocortical cell line (H295R). Based on the compelling MSI2 expression pattern, we set out to determine the role of MSI2 on aldosterone production. Depletion of MSI2 using siRNA led to significantly lower aldosterone levels in H295R cells stimulated with AngII. We also employed an orthogonal loss-of-function approach by co-treating cells with AngII and increasing concentrations of Ro-08-2750 (Ro), a direct and selective inhibitor of MSI2-RNA interactions. Ro inhibited aldosterone production in a dose-dependent manner at 1 µM with almost complete inhibition at 5 µM. The molecular mechanism by which MSI2 regulates target RNA translation and/or decay is unknown. Moreover, whether MSI2 acts as a repressor or activator appears to be context dependent. Our goal is to determine the precise molecular mechanism by which MSI2 promotes aldosterone production. Specifically, we will identify MSI2 targets, temporally resolved consequences of MSI2 inhibition, and protein interaction partners. This work will impact our understanding of fundamental principles of RBP-mediated regulation, as well as novel regulatory mechanisms underlying human steroid hormone synthesis. Indeed, Ro (or further optimized compounds) may represent new therapeutic avenues for adrenal disease.

Coiled-coil domain-containing protein-124 (Ccdc124) is a novel RNA binding factor up-regulated in endometrial, ovarian, and urinary bladder cancers

2021 ◽  
pp. 1-16
Author(s):  
Özge Arslan ◽  
Neşe Karadağ Soylu ◽  
Pelin Telkoparan Akıllılar ◽  
Uygar H. Tazebay
Keyword(s):  
Urinary Bladder ◽  
Rna Binding ◽  
Functional Characterization ◽  
Coiled Coil ◽  
Protein Accumulation ◽  
Mrna Levels ◽  
Aberrant Expression ◽  
Mrna Targets ◽  
Coiled Coil Domain ◽  
Binding Factor

BACKGROUND: Coiled-coil domain containing protein-124 (Ccdc124) is a putative mRNA-binding factor associated with cell division, and ribosome biology. Previous reports mentioned an up-regulation of CCDC124 gene in cancer, and listed its mRNA in a molecular prognostic signature in breast cancer. OBJECTIVES: Establishing RNA-binding characteristics of Ccdc124 for a better molecular functional characterization, and carrying-out retrospective studies in order to evaluate its aberrant expression in human cancer samples from various tissue origins. METHODS: Bioinformatics calculations followed by RIP and RNA-seq experiments were performed to investigate mRNA targets of Ccdc124. Quantitative studies on arrays of cDNAs from different cancers and IHC assays on tissue arrays were used to assess CCDC124 expression levels in cancers. RESULTS: Ccdc124 was characterized as an RNA-binding protein (RBP) interacting with various mRNAs. CCDC124 mRNA levels were high in tumors, with a particular up-regulation in cancers from esophagus, adrenal gland, endometrium, liver, ovary, thyroid, and urinary bladder. IHC assays indicated strong Ccdc124 positivity in endometrial (95.4%), urinary bladder (68.4%), and ovarian cancers (86.8%). CONCLUSION: Ccdc124 is a cytokinesis related RBP interacting with various mRNAs. CCDC124 mRNA over-expression and an accompanied increase in Ccdc124 protein accumulation was reported in cancers, indicating this RBP as a novel cancer cell marker.

scholarly journals Elevated METTL16 expression in PDAC confers synthetic lethality to PARP inhibition by antagonizing MRE11-mediated DNA end resection

2021 ◽  
Author(s):  
Xiangyu Zeng ◽  
Fei Zhao ◽  
Jake Kloeber ◽  
Rajashree Deshpande ◽  
Georges Mer ◽  
...  
Keyword(s):  
Rna Binding ◽  
Synthetic Lethality ◽  
Parp Inhibitor ◽  
Parp Inhibitors ◽  
Genetic Alterations ◽  
Ductal Adenocarcinoma ◽  
Parp Inhibition ◽  
Dependent Manner ◽  
Dna End Resection ◽  
End Resection

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Characterization of the recurrent genetic alterations in PDAC will yield improved understanding and therapies for this disease. Here, we report that PDAC patients with elevated expression of METTL16, one of the “writers” of RNA N6-methyladenosine (m6A) modification, may benefits from poly (ADP-ribose) polymerase (PARP) inhibitor treatment. Mechanistically, METTL16 interacts with MRE11 in an RNA-dependent manner; and, this interaction inhibits MRE11’s exonuclease activity in a methyltransferase-independent manner, thereby repressing DNA end resection. Upon DNA damage, ATM phosphorylates METTL16 at Ser419 within its C terminus, resulting in METTL16 conformational change and autoinhibition of its RNA binding. This dissociates the METTL16-RNA-MRE11 complex and releases inhibition of MRE11. Concordantly, PDAC cells with high METTL16 expression levels show increased sensitivity to PARP inhibitors, especially when combined with gemcitabine. Thus, our findings have revealed a role for METTL16 in homologous recombination repair and suggest that combination of PARP inhibitors with gemcitabine could be an effective treatment strategy for PDAC patients with high METTL16 expression.

scholarly journals The Nuclear Exosome Contributes to Autogenous Control of NAB2 mRNA Levels

2005 ◽  
Vol 25 (5) ◽  
pp. 1577-1585 ◽  
Author(s):  
Kelly M. Roth ◽  
Maria K. Wolf ◽  
Marie Rossi ◽  
J. Scott Butler
Keyword(s):  
Rna Processing ◽  
Untranslated Region ◽  
Rna Binding ◽  
Noncoding Rnas ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Autogenous Control ◽  
Regulatory Circuit ◽  
Mrna Surveillance ◽  
Nuclear Exosome

ABSTRACT The RNA-processing exosome is a complex of riboexonucleases required for 3′-end formation of some noncoding RNAs and for the degradation of mRNAs in eukaryotes. The nuclear form of the exosome functions in an mRNA surveillance pathway that retains and degrades improperly processed precursor mRNAs within the nucleus. We report here that the nuclear exosome controls the level of NAB2 mRNA, encoding the nuclear poly(A)+-RNA-binding protein Nab2p. Mutations affecting the activity of the nuclear, but not the cytoplasmic, exosome cause an increase in the amount of NAB2 mRNA. Cis- and trans-acting mutations that inhibit degradation by the nuclear-exosome subunit Rrp6p result in elevated levels of NAB2 mRNA. Control of NAB2 mRNA levels occurs posttranscriptionally and requires a sequence of 26 consecutive adenosines (A26) in the NAB2 3′ untranslated region, which represses NAB2 3′-end formation and sensitizes the transcript to degradation by Rrp6p. Analysis of NAB2 mRNA levels in a nab2-1 mutant and in the presence of excess Nab2p indicates that Nab2p activity negatively controls NAB2 mRNA levels in an A26- and Rrp6p-dependent manner. These findings suggest a novel regulatory circuit in which the nuclear exosome controls the level of NAB2 mRNA in response to changes in the activity of Nab2 protein.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

Grape Seed Proanthocyanidins Protect N2a Cells against Ischemic Injury via Endoplasmic Reticulum Stress and Mitochondrial-associated Pathways

2019 ◽  
Vol 18 (4) ◽  
pp. 334-341 ◽  
Author(s):  
Kun Fu ◽  
Liqiang Chen ◽  
Lifeng Miao ◽  
Yan Guo ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Cell Viability ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Grape Seed ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Grape Seed Proanthocyanidins ◽  
N2a Cells ◽  
Caspase 12

Background/Objective: Grape seed proanthocyanidins (GSPs) are a group of polyphenolic bioflavonoids, which possess a variety of biological functions and pharmacological properties. We studied the neuroprotective effects of GSP against oxygen-glucose deprivation/reoxygenation (OGD/R) injury and the potential mechanisms in mouse neuroblastoma N2a cells. Methods: OGD/R was conducted in N2a cells. Cell viability was evaluated by CCK-8 and LDH release assay. Apoptosis was assessed by TUNEL staining and flow cytometry. Protein levels of cleaved caspase-3, Bax and Bcl-2 were detected by Western blotting. CHOP, GRP78 and caspase-12 mRNA levels were assessed by real-time PCR. JC-1 dying was used to detect mitochondrial membrane potential. ROS levels, activities of endogenous antioxidant enzymes and ATP production were examined to evaluate mitochondrial function. Results: GSP increased cell viability after OGD/R injury in a dose-dependent manner. Furthermore, GSP inhibited cell apoptosis, reduced the mRNA levels of CHOP, GRP78 and caspase-12 (ER stressassociated genes), restored mitochondrial membrane potential and ATP generation, improved activities of endogenous anti-oxidant ability (T-AOC, GXH-Px, and SOD), and decreased ROS level. Conclusion: Our findings suggest that GSP can protect N2a cells from OGD/R insult. The mechanism of anti-apoptotic effects of GSP may involve attenuating ER stress and mitochondrial dysfunction.

Sign in / Sign up

Export Citation Format

Share Document