Biogenesis and functions of aminocarboxypropyluridine in tRNA

AbstractTransfer (t)RNAs contain a wide variety of post-transcriptional modifications, which play critical roles in tRNA stability and functions. 3-(3-amino-3-carboxypropyl)uridine (acp3U) is a highly conserved modification found in variable- and D-loops of tRNAs. Biogenesis and functions of acp3U have not been extensively investigated. Using a reverse-genetic approach supported by comparative genomics, we find here that the Escherichia coli yfiP gene, which we rename tapT (tRNA aminocarboxypropyltransferase), is responsible for acp3U formation in tRNA. Recombinant TapT synthesizes acp3U at position 47 of tRNAs in the presence of S-adenosylmethionine. Biochemical experiments reveal that acp3U47 confers thermal stability on tRNA. Curiously, the ΔtapT strain exhibits genome instability under continuous heat stress. We also find that the human homologs of tapT, DTWD1 and DTWD2, are responsible for acp3U formation at positions 20 and 20a of tRNAs, respectively. Double knockout cells of DTWD1 and DTWD2 exhibit growth retardation, indicating that acp3U is physiologically important in mammals.

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RNA-binding proteins and circadian rhythms in Arabidopsis thaliana

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2001.0964 ◽

2001 ◽

Vol 356 (1415) ◽

pp. 1755-1759 ◽

Cited By ~ 28

Author(s):

Dorothee Staiger

Keyword(s):

Arabidopsis Thaliana ◽

Feedback Loop ◽

Rna Binding ◽

Rna Binding Proteins ◽

Transcriptional Level ◽

Reverse Genetic ◽

Genetic Approach ◽

Circadian Oscillators ◽

Clock Proteins ◽

Reverse Genetic Approach

An Arabidopsis transcript preferentially expressed at the end of the daily light period codes for the RNA–binding protein At GRP7. A reverse genetic approach in Arabidopsis thaliana has revealed its role in the generation of circadian rhythmicity: At GRP7 is part of a negative feedback loop through which it influences the oscillations of its own transcript. Biochemical and genetic experiments indicate a mechanism for this autoregulatory circuit: At grp7 gene transcription is rhythmically activated by the circadian clock during the day. The At GPR7 protein accumulates with a certain delay and represses further accumulation of its transcript, presumably at the post–transcriptional level. In this respect, the At GRP7 feedback loop differs from known circadian oscillators in the fruitfly Drosophila and mammals based on oscillating clock proteins that repress transcription of their own genes with a 24 h rhythm. It is proposed that the At GRP7 feedback loop may act within an output pathway from the Arabidopsis clock.

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Structural and phenotypic analysis of Chikungunya virus RNA replication elements

Nucleic Acids Research ◽

10.1093/nar/gkz640 ◽

2019 ◽

Vol 47 (17) ◽

pp. 9296-9312 ◽

Cited By ~ 5

Author(s):

Catherine Kendall ◽

Henna Khalid ◽

Marietta Müller ◽

Dominic H Banda ◽

Alain Kohl ◽

...

Keyword(s):

Rational Design ◽

Chikungunya Virus ◽

Rna Replication ◽

Host Cells ◽

Genome Replication ◽

Reverse Genetic ◽

Genetic Approach ◽

Phenotypic Analysis ◽

Efficient Replication ◽

Reverse Genetic Approach

Abstract Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.

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A reverse genetic approach for generating gene replacement mutants in Ustilago maydis

Molecular Genetics and Genomics ◽

10.1007/s00438-004-1067-8 ◽

2004 ◽

Vol 272 (4) ◽

pp. 488-488 ◽

Cited By ~ 110

Author(s):

A. Brachmann ◽

J. König ◽

C. Julius ◽

M. Feldbrügge

Keyword(s):

Ustilago Maydis ◽

Gene Replacement ◽

Reverse Genetic ◽

Genetic Approach ◽

Reverse Genetic Approach

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The Reverse Genetic Approach to the Etiology of Schizophrenia

Search for the Causes of Schizophrenia ◽

10.1007/978-3-642-74881-3_12 ◽

1990 ◽

pp. 144-170

Author(s):

L. E. DeLisi ◽

M. Lovett

Keyword(s):

Reverse Genetic ◽

Genetic Approach ◽

Reverse Genetic Approach

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A reverse genetic approach identifies an ancestral frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds

Genetics Selection Evolution ◽

10.1186/s12711-016-0232-y ◽

2016 ◽

Vol 48 (1) ◽

Cited By ~ 13

Author(s):

Pauline Michot ◽

Sabine Chahory ◽

Andrew Marete ◽

Cécile Grohs ◽

Dimitri Dagios ◽

...

Keyword(s):

Retinal Degeneration ◽

Frameshift Mutation ◽

Reverse Genetic ◽

Genetic Approach ◽

Cattle Breeds ◽

European Cattle ◽

Reverse Genetic Approach

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Establishing RNA Interference as a Reverse-Genetic Approach for Gene Functional Analysis in Protoplasts

PLANT PHYSIOLOGY ◽

10.1104/pp.108.130260 ◽

2008 ◽

Vol 149 (2) ◽

pp. 642-652 ◽

Cited By ~ 61

Author(s):

Zhiyang Zhai ◽

Thanwalee Sooksa-nguan ◽

Olena K. Vatamaniuk

Keyword(s):

Functional Analysis ◽

Rna Interference ◽

Reverse Genetic ◽

Genetic Approach ◽

Gene Functional Analysis ◽

Reverse Genetic Approach

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Aminopeptidases and dipeptidyl-peptidases secreted by the dermatophyte Trichophyton rubrum

Microbiology ◽

10.1099/mic.0.27484-0 ◽

2005 ◽

Vol 151 (1) ◽

pp. 145-155 ◽

Cited By ~ 50

Author(s):

Michel Monod ◽

Barbara Léchenne ◽

Olivier Jousson ◽

Daniela Grand ◽

Christophe Zaugg ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Trichophyton Rubrum ◽

Hydrolytic Activity ◽

Cdna Libraries ◽

Sole Nitrogen Source ◽

Reverse Genetic ◽

Genetic Approach ◽

Leucine Aminopeptidases ◽

Dipeptidyl Peptidases ◽

Reverse Genetic Approach

The nature of secreted aminopeptidases in Trichophyton rubrum was investigated by using a reverse genetic approach. T. rubrum genomic and cDNA libraries were screened with Aspergillus spp. and Saccharomyces cerevisiae aminopeptidase genes as the probes. Two leucine aminopeptidases, ruLap1 and ruLap2, and two dipeptidyl-peptidases, ruDppIV and ruDppV, were characterized and compared to orthologues secreted by Aspergillus fumigatus using a recombinant protein from Pichia pastoris. RuLap1 is a 33 kDa nonglycosylated protein, while ruLap2 is a 58–65 kDa glycoprotein. The hydrolytic activity of ruLap1, ruLap2 and A. fumigatus orthologues showed various preferences for different aminoacyl-7-amido-4-methylcoumarin substrates, and various sensitivities to inhibitors and cations. ruDppIV and ruDppV showed similar activities to A. fumigatus orthologues. In addition to endopeptidases, the four aminopeptidases ruLap1, ruLap2, ruDppIV and ruDppV were produced by T. rubrum in a medium containing keratin as the sole nitrogen source. Synergism between endo- and exopeptidases is likely to be essential for dermatophyte virulence, since these fungi grow only in keratinized tissues.

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