scholarly journals Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen

2021 ◽  
Vol 14 (1) ◽  
pp. 11-33
Author(s):  
Sui Sawaguchi ◽  
Kenji Tago ◽  
Hiroaki Oizumi ◽  
Katsuya Ohbuchi ◽  
Masahiro Yamamoto ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Mtor Signaling ◽  
Marker Protein ◽  
Wild Type ◽  
Mutant Proteins ◽  
Oligodendroglial Cell ◽  
Hypomyelinating Leukodystrophy ◽  
In Cells

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.

scholarly journals Casein kinase II is required for efficient transcription by RNA polymerase III.

1996 ◽  
Vol 16 (3) ◽  
pp. 892-898 ◽  
Author(s):  
D J Hockman ◽  
M C Schultz
Keyword(s):  
Protein Kinase ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Rna Polymerases ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Polymerase Iii

Casein kinase II (CKII) is a ubiquitous and highly conserved serine/threonine protein kinase found in the nucleus and cytoplasm of most cells. Using a combined biochemical and genetic approach in the yeast Saccharomyces cerevisiae, we assessed the role of CKII in specific transcription by RNA polymerases I, II, and III. CKII is not required for basal transcription by RNA polymerases I and II but is important for polymerase III transcription. Polymerase III transcription is high in extracts with normal CKII activity but low in extracts from a temperature-sensitive mutant that has decreased CKII activity due to a lesion in the enzyme's catalytic alpha' subunit. Polymerase III transcription of 5S rRNA and tRNA templates in the temperature-sensitive extract is rescued by purified, wild-type CKII. An inhibitor of CKII represses polymerase III transcription in wild-type extract, and this repression is partly overcome by supplementing reaction mixtures with active CKII. Finally, we show that polymerase III transcription in vivo is impaired when CKII is inactivated. Our results demonstrate that CKII, an oncogenic protein kinase previously implicated in cell cycle and growth control, is required for high-level transcription by RNA polymerase III.

scholarly journals Novel Small-Molecule Inhibitors of RNA Polymerase III

2003 ◽  
Vol 2 (2) ◽  
pp. 256-264 ◽  
Author(s):  
Liping Wu ◽  
Jing Pan ◽  
Vala Thoroddsen ◽  
Deborah R. Wysong ◽  
Ronald K. Blackman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Rna Polymerase Iii ◽  
Wild Type ◽  
Biochemical Assays ◽  
Transcription In Vitro ◽  
Pol Iii

ABSTRACT A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.

Polyadenylation of Sine Transcripts Generated by RNA Polymerase III Dramatically Prolongs Their Lifetime in Cells

2020 ◽  
Vol 54 (1) ◽  
pp. 67-74 ◽  
Author(s):  
I. G. Ustyantsev ◽  
K. A. Tatosyan ◽  
D. V. Stasenko ◽  
N. Y. Kochanova ◽  
O. R. Borodulina ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Polymerase Iii ◽  
In Cells

Proteasome Inhibitors Restore to Normal the Decreased Levels of Protein Expression and Nucleolar Localization of Various Mutant Ribosomal S19 Proteins Identified in DBA Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 185-185 ◽  
Author(s):  
Aurore Cretien ◽  
Alexis Proust ◽  
Hanna Gazda ◽  
Jorg Meerpohl ◽  
Charlotte Marie Niemeyer ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Protein Expression ◽  
Proteasome Inhibitors ◽  
Nucleolar Localization ◽  
Wild Type ◽  
Mutant Proteins ◽  
Expression Levels ◽  
Type Protein ◽  
Wild Type Protein ◽  
Proteosomal Degradation

Abstract Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. We have previously shown that several RPS19 mutant proteins (V15F, InsAG36, W33stop, Y48stop, R56stop, M75stop, R94stop, 274del31, InsG238, G127Q and L131P) exhibit decreased levels of protein expression and do not localize to the nucleolus like the wild type protein in transfected Cos-7 cells. In contrast, other mutants (W52C, T55M, R56Q, R62W, 24Del18, G120S) exhibit normal levels of protein expression and normal nucleolar localization. We hypothesized that decreased levels of expression of mutant proteins such as V15F, G127Q, and L131P may be due to proteosomal degradation. In order to validate our hypothesis, we analyzed the effects of two proteasome inhibitors (MG132 and lactacystin) on mutant RPS19 protein expression levels and their subcellular localization. Following treatment with proteosome inhibitors, the mutant proteins with missense mutations (V15F, G127Q and L131P) were expressed at levels similar to that of wild type protein and localized in the nucleolus. Similarly, proteasome inhibitors also restored the expression levels and normal subcellular localization to RPS19 with non-sense mutations (InsG238, R94stop, and 274del31) that resulted in the translation of RPS19 protein with at least 80 aminoacids. In marked contrast, proteosome inhibitors failed to restore the expression levels of RPS19 with the non-sense mutants that led to synthesis of shortened proteins (InsAG36, W33stop, Y48stop, R56stop, M75stop). Even in the presence of proteosome inhibitors we noted a dramatic decrease in the levels of expression of these mutant proteins and proteins expressed were localized in the cytoplasm. Our findings imply an important role for proteosomal degradation pathway in regulating the expression levels of RPS19. They further suggest that proteasome inhibitors could be considered as a potential treatment for some steroid resistant DBA affected individuals with specific RPS19 mutations.

scholarly journals MAF1 is a Chronic Repressor of RNA Polymerase III Transcription in the Mouse

2019 ◽  
Author(s):  
Nicolas Bonhoure ◽  
Viviane Praz ◽  
Robyn D. Moir ◽  
Gilles Willemin ◽  
François Mange ◽  
...  
Keyword(s):  
Energy Expenditure ◽  
Rna Polymerase ◽  
Ribosomal Proteins ◽  
Rna Polymerase Iii ◽  
Mrna Levels ◽  
Wild Type ◽  
Pol Iii ◽  
The Impact ◽  
Global Translation ◽  
Modest Effect

AbstractMaf1-/- mice are lean, obesity-resistant and metabolically inefficient. Their increased energy expenditure is thought to be driven by a futile RNA cycle that reprograms metabolism to meet an increased demand for nucleotides stemming from the deregulation of RNA polymerase (pol) III transcription. Metabolic changes consistent with this model have been reported in both fasted and refed mice, however the impact of the fasting-refeeding-cycle on pol III function has not been examined. Here we show that changes in pol III occupancy in the liver of fasted versus refed wild-type mice are largely confined to low and intermediate occupancy genes; high occupancy genes are unchanged. However, in Maf1-/- mice, pol III occupancy of the vast majority of active loci in liver and the levels of specific precursor tRNAs in this tissue and other organs are higher than wild-type in both fasted and refed conditions. Thus, MAF1 functions as a chronic repressor of active pol III loci and can modulate transcription under different conditions. Our findings support the futile RNA cycle hypothesis, elaborate the mechanism of pol III repression by MAF1 and demonstrate a modest effect of MAF1 on global translation via reduced mRNA levels and translation efficiencies for several ribosomal proteins.

scholarly journals Differential Utilization of TATA Box-binding Protein (TBP) and TBP-related Factor 1 (TRF1) at Different Classes of RNA Polymerase III Promoters

2013 ◽  
Vol 288 (38) ◽  
pp. 27564-27570 ◽  
Author(s):  
Neha Verma ◽  
Ko-Hsuan Hung ◽  
Jin Joo Kang ◽  
Nermeen H. Barakat ◽  
William E. Stumph
Keyword(s):  
Rna Polymerase ◽  
Binding Protein ◽  
Rna Polymerase Iii ◽  
Tata Box ◽  
Related Factor ◽  
U6 Snrna ◽  
Polymerase Iii ◽  
Tata Box Binding Protein ◽  
In Cells

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459–469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells

2008 ◽  
Vol 294 (1) ◽  
pp. G58-G67 ◽  
Author(s):  
Tatehiro Kagawa ◽  
Norihito Watanabe ◽  
Kaori Mochizuki ◽  
Asano Numari ◽  
Yoshie Ikeno ◽  
...  
Keyword(s):  
Protein Expression ◽  
Apical Membrane ◽  
Bile Salt Export Pump ◽  
Transport Activity ◽  
Wild Type ◽  
Phenotypic Difference ◽  
Mutant Proteins ◽  
Phenotypic Differences ◽  
Benign Recurrent Intrahepatic Cholestasis ◽  
Taurocholate Transport

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.

scholarly journals A Putative ECF σ Factor Gene, rpoI, Regulates Siderophore Production in Rhizobium leguminosarum

1999 ◽  
Vol 12 (11) ◽  
pp. 994-999 ◽  
Author(s):  
Kay H. Yeoman ◽  
Alex G. May ◽  
Nicola G. deLuca ◽  
Daniel B. Stuckey ◽  
Andrew W. B. Johnston
Keyword(s):  
Rna Polymerase ◽  
Gene Product ◽  
Rhizobium Leguminosarum ◽  
Siderophore Production ◽  
Wild Type ◽  
Factor Gene ◽  
Fe Uptake ◽  
Σ Factor ◽  
Type Strains ◽  
In Cells

A cloned Rhizobium leguminosarum gene, termed rpoI, when transferred to wild-type strains, caused overproduction of the siderophore vicibactin. An rpoI mutant was defective in Fe uptake but was unaffected in symbiotic N2 fixation. The RpoI gene product was similar in sequence to extra-cytoplasmic σ factors of RNA polymerase. Transcription of rpoI was reduced in cells grown in medium that was replete with Fe.

scholarly journals Calbindin-D28kin the Brain Influences the Expression of Cellular Prion Protein

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Yeong-Min Yoo ◽  
Eui-Bae Jeung
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Expression ◽  
Prion Protein ◽  
Calcium Transport ◽  
Transport Proteins ◽  
Cellular Prion Protein ◽  
Marker Protein ◽  
Wild Type ◽  
Calbindin D9k ◽  
The Brain

The phenotypes ofcalbindin-D9k- (CaBP-9k-) knockout (KO),calbindin-D28k- (CaBP-28k-) KO, andCaBP-9k/28k-KO mice are similar to those of wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we investigated the expression of cellular prion protein (PrPC) in the brains ofCaBP-9k-,CaBP-28k-, andCaBP-9k/28k-KO mice. PrPCexpression was significantly upregulated in the brain of all three strains. Levels of phospho-Akt (Ser473) and phospho-Bad (Ser136) were significantly elevated, but those of phospho-ERK and phospho-Bad (Ser155 and 112) were significantly reduced in the brains ofCaBP-9k-,CaBP-28k-, andCaBP-9k/28k-KO mice. The expressions of the Bcl-2, p53, Bax, Cu/Zn-SOD, and Mn-SOD proteins were decreased in the brains of all KO mice. Expression of the endoplasmic reticulum marker protein BiP/GRP78 was decreased, and that of the CHOP protein was increased in the brains of those KO mice. To identify the roles ofCaBP-28k, we transfected PC12 cells with siRNA forCaBP-28kand found increased expression of the PrPCprotein compared to the levels in control cells. These results suggest thatCaBP-28kexpression may regulate PrPCprotein expression and these mice may be vulnerable to the influence of prion disease.

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