scholarly journals An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Eduarde Rohner ◽  
Nevin Witman ◽  
Jesper Sohlmer ◽  
Erwin De Genst ◽  
William E. Louch ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Transcriptional Profiling ◽  
Embryonic Stem ◽  
Assay System ◽  
Null Mutation ◽  
Molecular Pathways ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Fourfold Increase

Abstract Background The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. Methods A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. Results Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. Conclusion Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.

scholarly journals RNA Sequencing Reveals Transcriptomic Changes in Tobacco (Nicotiana Tabacum) Following NtCPS2 Knockout

2020 ◽  
Author(s):  
Shixiao Xu ◽  
Lingxiao He ◽  
Huabing Liu ◽  
Changhe Cheng ◽  
Dongfang Cai ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Rna Sequencing ◽  
Gene Knockout ◽  
Glandular Trichome ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Tobacco Plants ◽  
Genes Encoding ◽  
Plant Pathogen Interactions

Abstract Background: Amber-like compounds form in tobacco (Nicotiana tabacum) during leaf curing and impact aromatic quality. In particular, cis-abienol, a polycyclic labdane-related diterpenoid, is of research interest as a precursor of these compounds. Glandular trichome cells specifically express copalyl diphosphate synthase (NtCPS2) at high levels in tobacco, which, together with NtABS, are major regulators of cis-abienol biosynthesis in tobacco. Results: To identify the genes involved in the biosynthesis of cis-abienol in tobacco, we constructed transgenic tobacco lines based on an NtCPS2 gene-knockout model, using CRISPR/Cas9 genome-editing technology to inhibit NtCPS2 function in vitro. In mutant plants, cis-abienol and labdene-diol contents decreased, whereas gibberellin and abscisic acid (ABA) contents increased compared with those in wild-type tobacco plants. RNA sequencing analysis revealed the presence of 9,514 differentially expressed genes (DEGs; 4,279 upregulated, 5,235 downregulated) when the leaves of wild-type and NtCPS2-knockout tobacco plants were screened. Among these DEGs, the genes encoding cis-abienol synthase, ent-kaurene oxidase, auxin/ABA-related proteins, and transcription factors were found to be involved in various biological and physiochemical processes, including diterpenoid biosynthesis, plant hormone signal transduction, plant-pathogen interactions, etc. Conclusions: Our findings provide clues to the molecular regulatory mechanism underlying NtCPS2 activity, allowing for a better understanding of the interactions among related genes in tobacco.

scholarly journals RNA sequencing reveals transcriptomic changes in tobacco (Nicotiana tabacum) following NtCPS2 knockdown

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Lingxiao He ◽  
Huabing Liu ◽  
Changhe Cheng ◽  
Min Xu ◽  
Lei He ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Rna Sequencing ◽  
Glandular Trichome ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Tobacco Plants ◽  
Genes Encoding ◽  
Plant Pathogen Interactions ◽  
Transcriptomic Changes

Abstract Background Amber-like compounds form in tobacco (Nicotiana tabacum) during leaf curing and impact aromatic quality. In particular, cis-abienol, a polycyclic labdane-related diterpenoid, is of research interest as a precursor of these compounds. Glandular trichome cells specifically express copalyl diphosphate synthase (NtCPS2) at high levels in tobacco, which, together with NtABS, are major regulators of cis-abienol biosynthesis in tobacco. Results To identify the genes involved in the biosynthesis of cis-abienol in tobacco, we constructed transgenic tobacco lines based on an NtCPS2 gene-knockdown model using CRISPR/Cas9 genome-editing technology to inhibit NtCPS2 function in vitro. In mutant plants, cis-abienol and labdene diol contents decreased, whereas the gibberellin and abscisic acid (ABA) contents increased compared with those in wild-type tobacco plants. RNA sequencing analysis revealed the presence of 9514 differentially expressed genes (DEGs; 4279 upregulated, 5235 downregulated) when the leaves of wild-type and NtCPS2-knockdown tobacco plants were screened. Among these DEGs, the genes encoding cis-abienol synthase, ent-kaurene oxidase, auxin/ABA-related proteins, and transcription factors were found to be involved in various biological and physiochemical processes, including diterpenoid biosynthesis, plant hormone signal transduction, and plant-pathogen interactions. Conclusions The present study provides insight into the unique transcriptome profile of NtCPS2 knockdown tobacco, allowing for a better understanding of the biosynthesis of cis-abienol in tobacco.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

scholarly journals Defining dysfunction due to loss of MECP2 in Rett Patient Brain

2021 ◽  
Author(s):  
E Korsakova ◽  
A Morales ◽  
T McDaniel ◽  
A Lund ◽  
B Cooper ◽  
...  
Keyword(s):  
Rett Syndrome ◽  
Metabolic Pathways ◽  
Transcriptional Profiling ◽  
Childhood Development ◽  
Wild Type ◽  
Aberrant Expression ◽  
Metabolic Genes ◽  
Female Brain ◽  
Response To Stress

AbstractRett Syndrome is characterized by a postnatal loss of neurophysiological function and regression of childhood development. Because the syndrome is X-linked and males with MECP2 mutations generally do not survive birth, the study of this syndrome has been complicated by the fact that in female brain, a portion of neurons express wild type MECP2, and another portion express a non-functional allele of MECP2. Therefore, bulk-RNA-sequencing of Rett brain is confounded by the presence of chimerism of neurons for functional MECP2 in neurons. We developed an approach that allows for single-nuclei transcriptional profiling of individual neurons and a direct comparison between neurons that express functional MECP2 with those that express the disease-causing allele. We found that mutant neurons from Rett brain show patterns of aberrant expression of synaptic and metabolic genes, both of which can be detected in in vitro models of Rett Syndrome. We used these resources to identify a role for POU2F1/OCT1 transcription factor in mediating the response to stress due to loss of MECP2, highlighting a potential key molecular regulator of stress in Rett neurons. Together, our new sorting approach enables us to highlight defective molecular and metabolic pathways in Rett brain neurons and suggests that in vitro models could serve as valuable tools to further study this syndrome and potentially for development of novel therapeutics.

scholarly journals TET-mediated 5-methylcytosine oxidation in tRNA promotes translation

2020 ◽  
pp. jbc.RA120.014226
Author(s):  
Hui Shen ◽  
Robert Jordan Ontiveros ◽  
Michael C Owens ◽  
Monica Yun Liu ◽  
Uday Ghanty ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Biological Function ◽  
Catalytic Domain ◽  
Embryonic Stem ◽  
Wild Type ◽  
Oxidation Activity ◽  
Tet Enzymes ◽  
Mediated Oxidation

Oxidation of 5-methylcytosine (5mC) in DNA by the Ten-eleven translocation (TET) family of enzymes is indispensable for gene regulation in mammals. More recently, evidence has emerged to support a biological function for TET-mediated m5C oxidation in messenger RNA. Here, we describe a previously uncharacterized role of TET-mediated m5C oxidation in transfer RNA (tRNAs). We found that the TET-mediated oxidation product 5-hydroxylmethylcytosine (hm5C) is specifically enriched in tRNA inside cells and that the oxidation activity of TET2 on m5C in tRNAs can be readily observed in vitro. We further observed that hm5C levels in tRNA were significantly decreased in Tet2 KO mouse embryonic stem cells (mESCs) in comparison to wild type mESCs. Reciprocally, induced expression of the catalytic domain of TET2 led to an obvious increase in hm5C and a decrease in m5C in tRNAs relative to uninduced cells. Strikingly, we also show that TET2-mediated m5C oxidation in tRNA promotes translation in vitro. These results suggest TET2 may influence translation through impacting tRNA methylation and reveal an unexpected role for TET enzymes in regulating multiple nodes of the central dogma.

scholarly journals Identification of RIFL, a novel adipocyte-enriched insulin target gene with a role in lipid metabolism

2012 ◽  
Vol 303 (3) ◽  
pp. E334-E351 ◽  
Author(s):  
Gang Ren ◽  
Ji Young Kim ◽  
Cynthia M. Smas
Keyword(s):  
Lipid Metabolism ◽  
Transcriptional Profiling ◽  
Serum Triglyceride ◽  
Transcript Level ◽  
Wild Type ◽  
New Genes ◽  
Brown Adipose ◽  
Genes Encoding ◽  
Triglyceride Content

To identify new genes that are important in fat metabolism, we utilized the Lexicon-Genentech knockout database of genes encoding transmembrane and secreted factors and whole murine genome transcriptional profiling data that we generated for 3T3-L1 in vitro adipogenesis. Cross-referencing null models evidencing metabolic phenotypes with genes induced in adipogenesis led to identification of a new gene, which we named RIFL (refeeding induced fat and liver). RIFL-null mice have serum triglyceride levels approximately one-third of wild type. RIFL transcript is induced >100-fold during 3T3-L1 adipogenesis and is also increased markedly during adipogenesis of murine and human primary preadipocytes. siRNA-mediated knockdown of RIFL during 3T3-L1 adipogenesis results in an ∼35% decrease in adipocyte triglyceride content. Murine RIFL transcript is highly enriched in white and brown adipose tissue and liver. Fractionation of WAT reveals that RIFL transcript is exclusive to adipocytes with a lack of expression in stromal-vascular cells. Nutritional and hormonal studies are consistent with a prolipogenic function for RIFL. There is evidence of an approximately eightfold increase in RIFL transcript level in WAT in ob/ob mice compared with wild-type mice. RIFL transcript level in WAT and liver is increased ∼80- and 12-fold, respectively, following refeeding of fasted mice. Treatment of 3T3-L1 adipocytes with insulin increases RIFL transcript ≤35-fold, whereas agents that stimulate lipolysis downregulate RIFL. Interestingly, the 198-amino acid RIFL protein is predicted to be secreted and shows ∼30% overall conservation with the NH2-terminal half of angiopoietin-like 3, a liver-secreted protein that impacts lipid metabolism. In summary, our data suggest that RIFL is an important new regulator of lipid metabolism.

scholarly journals Deficiency of the Stress Kinase P38α Results in Embryonic Lethality

2000 ◽  
Vol 191 (5) ◽  
pp. 859-870 ◽  
Author(s):  
Melanie Allen ◽  
Linne Svensson ◽  
Marsha Roach ◽  
John Hambor ◽  
John McNeish ◽  
...  
Keyword(s):  
Map Kinase ◽  
Es Cells ◽  
Interleukin 1 ◽  
Embryonic Stem ◽  
Wild Type ◽  
Antiinflammatory Drugs ◽  
Gene Encoding ◽  
Significant Difference ◽  
Induced Apoptosis

The mitogen-activated protein (MAP) kinase p38 is a key component of stress response pathways and the target of cytokine-suppressing antiinflammatory drugs (CSAIDs). A genetic approach was employed to inactivate the gene encoding one p38 isoform, p38α. Mice null for the p38α allele die during embryonic development. p38α1/− embryonic stem (ES) cells grown in the presence of high neomycin concentrations demonstrated conversion of the wild-type allele to a targeted allele. p38α−/− ES cells lacked p38α protein and failed to activate MAP kinase–activated protein (MAPKAP) kinase 2 in response to chemical stress inducers. In contrast, p38α1/+ ES cells and primary embryonic fibroblasts responded to stress stimuli and phosphorylated p38α, and activated MAPKAP kinase 2. After in vitro differentiation, both wild-type and p38α−/− ES cells yielded cells that expressed the interleukin 1 receptor (IL-1R). p38α1/+ but not p38α−/− IL-1R–positive cells responded to IL-1 activation to produce IL-6. Comparison of chemical-induced apoptosis processes revealed no significant difference between the p38α1/+ and p38α−/− ES cells. Therefore, these studies demonstrate that p38α is a major upstream activator of MAPKAP kinase 2 and a key component of the IL-1 signaling pathway. However, p38α does not serve an indispensable role in apoptosis.

GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 776-776
Author(s):  
Zhongfa Yang ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Target Genes ◽  
Embryonic Stem ◽  
Myeloid Cells ◽  
Transactivation Domain ◽  
Wild Type ◽  
Floxed Allele

Abstract GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.

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