InsP7is a small-molecule regulator of NUDT3-mediated mRNA decapping and processing-body dynamics

Regulation of enzymatic 5′ decapping of messenger RNA (mRNA), which normally commits transcripts to their destruction, has the capacity to dynamically reshape the transcriptome. For example, protection from 5′ decapping promotes accumulation of mRNAs into processing (P) bodies—membraneless, biomolecular condensates. Such compartmentalization of mRNAs temporarily removes them from the translatable pool; these repressed transcripts are stabilized and stored until P-body dissolution permits transcript reentry into the cytosol. Here, we describe regulation of mRNA stability and P-body dynamics by the inositol pyrophosphate signaling molecule 5-InsP7(5-diphosphoinositol pentakisphosphate). First, we demonstrate 5-InsP7inhibits decapping by recombinant NUDT3 (Nudix [nucleoside diphosphate linked moiety X]-type hydrolase 3) in vitro. Next, in intact HEK293 and HCT116 cells, we monitored the stability of a cadre of NUDT3 mRNA substrates following CRISPR-Cas9 knockout ofPPIP5Ks(diphosphoinositol pentakisphosphate 5-kinases type 1 and 2, i.e.,PPIP5KKO), which elevates cellular 5-InsP7levels by two- to threefold (i.e., within the physiological rheostatic range). ThePPIP5KKO cells exhibited elevated levels of NUDT3 mRNA substrates and increased P-body abundance. Pharmacological and genetic attenuation of 5-InsP7synthesis in the KO background reverted both NUDT3 mRNA substrate levels and P-body counts to those of wild-type cells. Furthermore, liposomal delivery of a metabolically resistant 5-InsP7analog into wild-type cells elevated levels of NUDT3 mRNA substrates and raised P-body abundance. In the context that cellular 5-InsP7levels normally fluctuate in response to changes in the bioenergetic environment, regulation of mRNA structure by this inositol pyrophosphate represents an epitranscriptomic control process. The associated impact on P-body dynamics has relevance to regulation of stem cell differentiation, stress responses, and, potentially, amelioration of neurodegenerative diseases and aging.

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High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽

10.1182/blood.v96.4.1517.h8001517_1517_1524 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1517-1524 ◽

Cited By ~ 1

Author(s):

Marjan J. T. Veuger ◽

M. Willy Honders ◽

Jim E. Landegent ◽

Roel Willemze ◽

Renée M. Y. Barge

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Messenger Rna ◽

Myeloid Leukemia ◽

Deoxycytidine Kinase ◽

Wild Type ◽

Healthy Donors ◽

Alternatively Spliced ◽

Acute Myeloid

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

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High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia

Blood ◽

10.1182/blood.v96.4.1517 ◽

2000 ◽

Vol 96 (4) ◽

pp. 1517-1524 ◽

Cited By ~ 57

Author(s):

Marjan J. T. Veuger ◽

M. Willy Honders ◽

Jim E. Landegent ◽

Roel Willemze ◽

Renée M. Y. Barge

Keyword(s):

T Cells ◽

Acute Myeloid Leukemia ◽

Messenger Rna ◽

Myeloid Leukemia ◽

Deoxycytidine Kinase ◽

Wild Type ◽

Healthy Donors ◽

Alternatively Spliced ◽

Acute Myeloid

Abstract Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML.

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Difference in Stability of the N-domain Underlies Distinct Intracellular Properties of the E1064A and H1069Q Mutants of Copper-transporting ATPase ATP7B

Journal of Biological Chemistry ◽

10.1074/jbc.m110.198101 ◽

2011 ◽

Vol 286 (18) ◽

pp. 16355-16362 ◽

Cited By ~ 24

Author(s):

Oleg Y. Dmitriev ◽

Ashima Bhattacharjee ◽

Sergiy Nokhrin ◽

Eva-Maria E. Uhlemann ◽

Svetlana Lutsenko

Keyword(s):

Phenotypic Variability ◽

Copper Metabolism ◽

Complete Loss ◽

Atp Binding ◽

Wild Type ◽

Helical Hairpin ◽

The Difference ◽

The Stability ◽

Golgi Network

Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu1064 and His1069 is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.

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TET-mediated 5-methylcytosine oxidation in tRNA promotes translation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014226 ◽

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.

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The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

Biochemical Journal ◽

10.1042/bj3120273 ◽

1995 ◽

Vol 312 (1) ◽

pp. 273-280 ◽

Cited By ~ 40

Author(s):

M Haraguchi ◽

S Yamashiro ◽

K Furukawa ◽

K Takamiya ◽

H Shiku ◽

...

Keyword(s):

Enzyme Activity ◽

Intracellular Localization ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Kinetics Analysis ◽

Glycosylation Sites ◽

In The Wild ◽

Polymerase Chain ◽

The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

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Mouse lysocardiolipin acyltransferase controls the development of hematopoietic and endothelial lineages during in vitro embryonic stem-cell differentiation

Blood ◽

10.1182/blood-2007-04-086827 ◽

2007 ◽

Vol 110 (10) ◽

pp. 3601-3609 ◽

Cited By ~ 25

Author(s):

Chengyan Wang ◽

Patrick W. Faloon ◽

Zhijia Tan ◽

Yaxin Lv ◽

Pengbo Zhang ◽

...

Keyword(s):

Cell Differentiation ◽

Messenger Rna ◽

Molecular Mechanisms ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryoid Bodies ◽

Embryonic Stem Cell Differentiation ◽

Hematopoietic Stem ◽

Endothelial Genes

Abstract The blast colony-forming cell (BL-CFC) was identified as an equivalent to the hemangioblast during in vitro embryonic stem (ES) cell differentiation. However, the molecular mechanisms underlying the generation of the BL-CFC remain largely unknown. Here we report the isolation of mouse lysocardiolipin acyltransferase (Lycat) based on homology to zebrafish lycat, a candidate gene for the cloche locus. Mouse Lycat is expressed in hematopoietic organs and is enriched in the Lin−C-Kit+Sca-1+ hematopoietic stem cells in bone marrow and in the Flk1+/hCD4+(Scl+) hemangioblast population in embryoid bodies. The forced Lycat transgene leads to increased messenger RNA expression of hematopoietic and endothelial genes as well as increased blast colonies and their progenies, endothelial and hematopoietic lineages. The Lycat small interfering RNA transgene leads to a decrease expression of hematopoietic and endothelial genes. An unbiased genomewide microarray analysis further substantiates that the forced Lycat transgene specifically up-regulates a set of genes related to hemangioblasts and hematopoietic and endothelial lineages. Therefore, mouse Lycat plays an important role in the early specification of hematopoietic and endothelial cells, probably acting at the level of the hemangioblast.

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Glycosylphosphatidylinositol (GPI) Proteins ofSaccharomyces cerevisiaeContain Ethanolamine Phosphate Groups on the α1,4-linked Mannose of the GPI Anchor

Journal of Biological Chemistry ◽

10.1074/jbc.m401873200 ◽

2004 ◽

Vol 279 (19) ◽

pp. 19614-19627 ◽

Cited By ~ 33

Author(s):

Isabella Imhof ◽

Isabelle Flury ◽

Christine Vionnet ◽

Carole Roubaty ◽

Diane Egger ◽

...

Keyword(s):

Side Chains ◽

Wild Type ◽

Genetic Manipulations ◽

Spontaneous Hydrolysis ◽

Lipid Precursor ◽

Gpi Proteins ◽

The Stability ◽

The One ◽

Ethanolamine Phosphate

In humans andSaccharomyces cerevisiaethe free glycosylphosphatidylinositol (GPI) lipid precursor contains several ethanolamine phosphate side chains, but these side chains had been found on the protein-bound GPI anchors only in humans, not yeast. Here we confirm that the ethanolamine phosphate side chain added by Mcd4p to the first mannose is a prerequisite for the addition of the third mannose to the GPI precursor lipid and demonstrate that, contrary to an earlier report, an ethanolamine phosphate can equally be found on the majority of yeast GPI protein anchors. Curiously, the stability of this substituent during preparation of anchors is much greater ingpi7Δsec18double mutants than in either single mutant or wild type cells, indicating that the lack of a substituent on the second mannose (caused by the deletion ofGPI7) influences the stability of the one on the first mannose. The phosphodiester-linked substituent on the second mannose, probably a further ethanolamine phosphate, is added to GPI lipids by endoplasmic reticulum-derived microsomesin vitrobut cannot be detected on GPI proteins of wild type cells and undergoes spontaneous hydrolysis in saline. Genetic manipulations to increase phosphatidylethanolamine levels ingpi7Δ cells by overexpression ofPSD1restore cell growth at 37 °C without restoring the addition of a substituent to Man2. The three putative ethanolamine-phosphate transferases Gpi13p, Gpi7p, and Mcd4p cannot replace each other even when overexpressed. Various models trying to explain how Gpi7p, a plasma membrane protein, directs the addition of ethanolamine phosphate to mannose 2 of the GPI core have been formulated and put to the test.

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Histone demethylase KDM4C controls tumorigenesis of glioblastoma by epigenetically regulating p53 and c-Myc

Cell Death and Disease ◽

10.1038/s41419-020-03380-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dong Hoon Lee ◽

Go Woon Kim ◽

Jung Yoo ◽

Sang Wu Lee ◽

Yu Hyun Jeon ◽

...

Keyword(s):

Tumor Suppressor Genes ◽

Therapeutic Strategy ◽

Target Gene ◽

Molecular Mechanisms ◽

Histone Demethylase ◽

Wild Type ◽

P53 Target Gene ◽

The Stability

AbstractGlioblastoma is the most lethal brain tumor and its pathogenesis remains incompletely understood. KDM4C is a histone H3K9 demethylase that contributes to epigenetic regulation of both oncogene and tumor suppressor genes and is often overexpressed in human tumors, including glioblastoma. However, KDM4C’s roles in glioblastoma and the underlying molecular mechanisms remain unclear. Here, we show that KDM4C knockdown significantly represses proliferation and tumorigenesis of glioblastoma cells in vitro and in vivo that are rescued by overexpressing wild-type KDM4C but not a catalytic dead mutant. KDM4C protein expression is upregulated in glioblastoma, and its expression correlates with c-Myc expression. KDM4C also binds to the c-Myc promoter and induces c-Myc expression. Importantly, KDM4C suppresses the pro-apoptotic functions of p53 by demethylating p53K372me1, which is pivotal for the stability of chromatin-bound p53. Conversely, depletion or inhibition of KDM4C promotes p53 target gene expression and induces apoptosis in glioblastoma. KDM4C may serve as an oncogene through the dual functions of inactivation of p53 and activation of c-Myc in glioblastoma. Our study demonstrates KDM4C inhibition as a promising therapeutic strategy for targeting glioblastoma.

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In vitro Protein Synthesis and Measurement of the Stability of Messenger RNA in the Blue-green Alga, Anabaena variabilis

Microbiology ◽

10.1099/00221287-81-1-47 ◽

2000 ◽

Vol 81 (1) ◽

pp. 47-58 ◽

Cited By ~ 7

Author(s):

C. K. Leach ◽

N. G. Carr

Keyword(s):

Amino Acids ◽

Green Alga ◽

Messenger Rna ◽

Antibiotic Sensitivity ◽

Anabaena Variabilis ◽

Blue Green Alga ◽

Peptide Formation ◽

A Cell ◽

The Stability

A cell-free preparation of the blue-green alga, Anabaena variabilis, that incorporates 14C-labelled amino acids into protein has been prepared and characterized. The activation of amino acids to amino acid transfer RNAs was characterized, and assembly of these into peptides has been described with respect to cofactor requirements and antibiotic sensitivity. The properties of these systems and the effect of antibiotics on them are similar to those of bacteria. Both natural and synthetic messenger RNA were effective in peptide formation. The kinetics of incorporation of [14C]uracil into RNA has been examined and the stability of labelled RNA from A. variabilis measured by radioactivity loss and by its role in directing peptide synthesis. The half-life of messenger RNA from this organism is approximately 12 min, and as such is comparable to that of bacteria when based upon the mean generation time of the respective organisms.

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Chromosome-Based Genetic Complementation System for Xylella fastidiosa

Applied and Environmental Microbiology ◽

10.1128/aem.00024-09 ◽

2009 ◽

Vol 75 (6) ◽

pp. 1679-1687 ◽

Cited By ~ 36

Author(s):

Ayumi Matsumoto ◽

Glenn M. Young ◽

Michele M. Igo

Keyword(s):

Dna Sequences ◽

Xylella Fastidiosa ◽

Cell Physiology ◽

Multiple Cloning Site ◽

Wild Type ◽

In Planta ◽

Catalase Peroxidase ◽

The Stability

ABSTRACT Xylella fastidiosa is a xylem-limited, gram-negative bacterium that causes Pierce's disease of grapevine. Here, we describe the construction of four vectors that facilitate the insertion of genes into a neutral site (NS1) in the X. fastidiosa chromosome. These vectors carry a colE1-like (pMB1) replicon and DNA sequences from NS1 flanking a multiple-cloning site and a resistance marker for one of the following antibiotics: chloramphenicol, erythromycin, gentamicin, or kanamycin. In X. fastidiosa, vectors with colE1-like (pMB1) replicons have been found to result primarily in the recovery of double recombinants rather than single recombinants. Thus, the ease of obtaining double recombinants and the stability of the resulting insertions at NS1 in the absence of selective pressure are the major advantages of this system. Based on in vitro and in planta characterizations, strains carrying insertions within NS1 are indistinguishable from wild-type X. fastidiosa in terms of growth rate, biofilm formation, and pathogenicity. To illustrate the usefulness of this system for complementation analysis, we constructed a strain carrying a mutation in the X. fastidiosa cpeB gene, which is predicted to encode a catalase/peroxidase, and showed that the sensitivity of this mutant to hydrogen peroxide could be overcome by the introduction of a wild-type copy of cpeB at NS1. Thus, this chromosome-based complementation system provides a valuable genetic tool for investigating the role of specific genes in X. fastidiosa cell physiology and virulence.

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