scholarly journals Structural and catalytic roles of the human 18S rRNA methyltransferases DIMT1 in ribosome assembly and translation

2020 ◽  
Vol 295 (34) ◽  
pp. 12058-12070 ◽  
Author(s):  
Hui Shen ◽  
Julian Stoute ◽  
Kathy Fange Liu

rRNA-modifying enzymes participate in ribosome assembly. However, whether the catalytic activities of these enzymes are important for the ribosome assembly and other cellular processes is not fully understood. Here, we report the crystal structure of WT human dimethyladenosine transferase 1 (DIMT1), an 18S rRNA N6,6-dimethyladenosine (m26,6A) methyltransferase, and results obtained with a catalytically inactive DIMT1 variant. We found that DIMT1+/− heterozygous HEK 293T cells have a significantly decreased 40S fraction and reduced protein synthesis but no major changes in m26,6A levels in 18S rRNA. Expression of a catalytically inactive variant, DIMT1-E85A, in WT and DIMT1+/− cells significantly decreased m26,6A levels in 18S rRNA, indicating a dominant-negative effect of this variant on m26,6A levels. However, expression of the DIMT1-E85A variant restored the defects in 40S levels. Of note, unlike WT DIMT1, DIMT1-E85A could not revert the defects in protein translation. We found that the differences between this variant and the WT enzyme extended to translation fidelity and gene expression patterns in DNA damage response pathways. These results suggest that the catalytic activity of DIMT1 is involved in protein translation and that the overall protein scaffold of DIMT1, regardless of the catalytic activity on m26,6A in 18S rRNA, is essential for 40S assembly.

2017 ◽  
Vol 28 (22) ◽  
pp. 3029-3042 ◽  
Author(s):  
Xiaoyan Zhu ◽  
Emiliya Poghosyan ◽  
Radhika Gopal ◽  
Yi Liu ◽  
Kristine S. Ciruelas ◽  
...  

Nucleoside diphosphate kinases (NDKs) play a central role in diverse cellular processes using the canonical NDK activity or alternative mechanisms that remain poorly defined. Our study of dimeric NDK5 in a flagellar motility control complex, the radial spoke (RS), has revealed new modalities. The flagella in Chlamydomonas ndk5 mutant were paralyzed, albeit only deficient in three RS subunits. RS morphology appeared severely changed in averaged cryo-electron tomograms, suggesting that NDK5 is crucial for the intact spokehead formation as well as RS structural stability. Intriguingly, ndk5’s flagella were also short, resembling those of an allelic spoke-less mutant. All ndk5’s phenotypes were rescued by expressions of NDK5 or a mutated NDK5 lacking the canonical kinase activity. Importantly, the mutated NDK5 that appeared fully functional in ndk5 cells elicited a dominant-negative effect in wild-type cells, causing paralyzed short flagella with hypophosphorylated, less abundant, but intact RSs, and accumulated hypophosphorylated NDK5 in the cell body. We propose that NDK5 dimer is an RS structural subunit with an additional mechanism that uses cross-talk between the two NDK monomers to accelerate phosphorylation-related assembly of RSs and entire flagella.


Genetics ◽  
1996 ◽  
Vol 144 (4) ◽  
pp. 1455-1462
Author(s):  
José L Barra ◽  
Mario R Mautino ◽  
Alberto L Rosa

eth-1r a thermosensitive allele of the Neurospora crassa S-adenosylmethionine (AdoMet) synthetase gene that confers ethionine resistance, has been cloned and sequenced. Replacement of an aspartic amino acid residue (D48 → N48), perfectly conserved in prokaryotic, fungal and higher eukaryotic AdoMet synthetases, was found responsible for both thermosensitivity and ethionine resistance conferred by eth-1r. Gene fusion constructs, designed to overexpress eth-1r in vivo, render transformant cells resistant to ethionine. Dominance of ethionine resistance was further demonstrated in eth-1  +/eth-1r partial diploids carrying identical gene doses of both alleles. Heterozygous eth-1  +/eth-1r cells have, at the same time, both the thermotolerance conferred by eth-1  + and the ethionine-resistant phenotype conferred by eth-1r. AdoMet levels and AdoMet synthetase activities were dramatically decreased in heterozygous eth-1  +/eth-1r cells. We propose that this negative effect exerted by eth-1r results from the in vivo formation of heteromeric eth-1  +/eth-1r AdoMet synthetase molecules.


Genetics ◽  
2002 ◽  
Vol 162 (2) ◽  
pp. 633-645 ◽  
Author(s):  
Guido Cuperus ◽  
David Shore

Abstract We previously described two classes of SIR2 mutations specifically defective in either telomeric/HM silencing (class I) or rDNA silencing (class II) in S. cerevisiae. Here we report the identification of genes whose protein products, when either overexpressed or directly tethered to the locus in question, can establish silencing in SIR2 class I mutants. Elevated dosage of SCS2, previously implicated as a regulator of both inositol biosynthesis and telomeric silencing, suppressed the dominant-negative effect of a SIR2-143 mutation. In a genetic screen for proteins that restore silencing when tethered to a telomere, we isolated ESC2 and an uncharacterized gene, (YOL017w), which we call ESC8. Both Esc2p and Esc8p interact with Sir2p in two-hybrid assays, and the Esc8p-Sir2 interaction is detected in vitro. Interestingly, Esc8p has a single close homolog in yeast, the ISW1-complex factor Ioc3p, and has also been copurified with Isw1p, raising the possibility that Esc8p is a component of an Isw1p-containing nucleosome remodeling complex. Whereas esc2 and esc8 deletion mutants alone have only marginal silencing defects, cells lacking Isw1p show a strong silencing defect at HMR but not at telomeres. Finally, we show that Esc8p interacts with the Gal11 protein, a component of the RNA pol II mediator complex.


Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.


Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 830
Author(s):  
Prasangi Rajapaksha ◽  
Isoiza Ojo ◽  
Ling Yang ◽  
Ankit Pandeya ◽  
Thilini Abeywansha ◽  
...  

The RND family efflux pump AcrAB-TolC in E. coli and its homologs in other Gram-negative bacteria are major players in conferring multidrug resistance to the cells. While the structure of the pump complex has been elucidated with ever-increasing resolution through crystallography and Cryo-EM efforts, the dynamic assembly process remains poorly understood. Here, we tested the effect of overexpressing functionally defective pump components in wild type E. coli cells to probe the pump assembly process. Incorporation of a defective component is expected to reduce the efflux efficiency of the complex, leading to the so called “dominant negative” effect. Being one of the most intensively studied bacterial multidrug efflux pumps, many AcrA and AcrB mutations have been reported that disrupt efflux through different mechanisms. We examined five groups of AcrB and AcrA mutants, defective in different aspects of assembly and substrate efflux. We found that none of them demonstrated the expected dominant negative effect, even when expressed at concentrations many folds higher than their genomic counterpart. The assembly of the AcrAB-TolC complex appears to have a proof-read mechanism that effectively eliminated the formation of futile pump complex.


Genes ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 875
Author(s):  
Karlijn Pellikaan ◽  
Geeske M. van Woerden ◽  
Lotte Kleinendorst ◽  
Anna G. W. Rosenberg ◽  
Bernhard Horsthemke ◽  
...  

Prader–Willi syndrome (PWS) is a rare genetic condition characterized by hypotonia, intellectual disability, and hypothalamic dysfunction, causing pituitary hormone deficiencies and hyperphagia, ultimately leading to obesity. PWS is most often caused by the loss of expression of a cluster of genes on chromosome 15q11.2-13. Patients with Prader–Willi-like syndrome (PWLS) display features of the PWS phenotype without a classical PWS genetic defect. We describe a 46-year-old patient with PWLS, including hypotonia, intellectual disability, hyperphagia, and pituitary hormone deficiencies. Routine genetic tests for PWS were normal, but a homozygous missense variant NM_003097.3(SNRPN):c.193C>T, p.(Arg65Trp) was identified. Single nucleotide polymorphism array showed several large regions of homozygosity, caused by high-grade consanguinity between the parents. Our functional analysis, the ‘Pipeline for Rapid in silico, in vivo, in vitro Screening of Mutations’ (PRiSM) screen, showed that overexpression of SNRPN-p.Arg65Trp had a dominant negative effect, strongly suggesting pathogenicity. However, it could not be confirmed that the variant was responsible for the phenotype of the patient. In conclusion, we present a unique homozygous missense variant in SNURF-SNRPN in a patient with PWLS. We describe the diagnostic trajectory of this patient and the possible contributors to her phenotype in light of the current literature on the genotype–phenotype relationship in PWS.


2021 ◽  
pp. 002203452199662
Author(s):  
J.T. Chen ◽  
C.H. Lin ◽  
H.W. Huang ◽  
Y.P. Wang ◽  
P.C. Kao ◽  
...  

Hereditary gingival fibromatosis (HGF) is a rare genetic disorder featured by nonsyndromic pathological overgrowth of gingiva. The excessive gingival tissues can cause dental, masticatory, and phonetic problems, which impose severe functional and esthetic burdens on affected individuals. Due to its high recurrent rate, patients with HGF have to undergo repeated surgical procedures of gingival resection, from childhood to adulthood, which significantly compromises their quality of life. Unraveling the genetic etiology and molecular pathogenesis of HGF not only gains insight into gingival physiology and homeostasis but also opens avenues for developing potential therapeutic strategies for this disorder. Recently, mutations in REST (OMIM *600571), encoding a transcription repressor, were reported to cause HGF (GINGF5; OMIM #617626) in 3 Turkish families. However, the functions of REST in gingival homeostasis and pathogenesis of REST-associated HGF remain largely unknown. In this study, we characterized 2 HGF families and identified 2 novel REST mutations, c.2449C>T (p.Arg817*) and c.2771_2793dup (p.Glu932Lysfs*3). All 5 mutations reported to date are nonsenses or frameshifts in the last exon of REST and would presumably truncate the protein. In vitro reporter gene assays demonstrated a partial or complete loss of repressor activity for these truncated RESTs. When coexpressed with the full-length protein, the truncated RESTs impaired the repressive ability of wild-type REST, suggesting a dominant negative effect. Immunofluorescent studies showed nuclear localization of overexpressed wild-type and truncated RESTs in vitro, indicating preservation of the nuclear localization signal in shortened proteins. Immunohistochemistry demonstrated a comparable pattern of ubiquitous REST expression in both epithelium and lamina propria of normal and HGF gingival tissues despite a reduced reactivity in HGF gingiva. Results of this study confirm the pathogenicity of REST truncation mutations occurring in the last exon causing HGF and suggest the pathosis is caused by an antimorphic (dominant negative) disease mechanism.


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