Pseudouridine: Still mysterious, but never a fake (uridine)!

Spenkuch, Felix; Motorin, Yuri; Helm, Mark

doi:10.4161/15476286.2014.992278

Cited by 172 publications

(198 citation statements)

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“…lanes 1-2 and 3-10) and, as for U6, these stops were not dependent onTgPUS1 (arrows, lanes 4 and 6, 8 and 10). These putative Ψ's are found in a region of U2 that is pseudouridylated in S. cerevisiae and humans (Spenkuch et al 2014).…”

Section: Pseudouridylation Of Spliceosomal Rnas Is Tgpus1-independentmentioning

confidence: 99%

“…One of the most abundant, ubiquitous, and highly conserved modifications is pseudouridine (psi or Ψ), an isomerization of uridine in which the C5 position, rather than the N1, is incorporated into the glycosidic bond (Cohn 1959(Cohn , 1960Scannell et al 1959;Yu and Allen 1959). This modification is found at high levels in rRNA in multiple organisms and is highly evolutionarily conserved at multiple sites in rRNA, tRNA, and spliceosomal RNA (Volkin and Cohn 1951;Spenkuch et al 2014;Li et al 2016). Most recently, with the advent of highthroughput sequencing tools, Ψ has also been found in mRNAs (Carlile et al 2014;Lovejoy et al 2014;Schwartz et al 2014;Li et al 2015).…”

Section: Introductionmentioning

confidence: 99%

“…These enzymes can be divided into RNA-independent PUSs, which recognize their targets based on a combination of sequence and secondary structure, and RNA-dependent PUSs, which recognize their targets with the help of a protein complex containing an H/ACA guide RNA that is complementary to the target sequence (Ofengand 2002;Hamma and Ferré-D'Amaré 2006). While RNA-independent PUSs target tRNAs, snRNAs, mRNAs, and prokaryotic rRNA, RNA-dependent PUSs target snRNAs, mRNAs, and eukaryotic rRNA (Spenkuch et al 2014;Safra et al 2017).…”

Section: Introductionmentioning

confidence: 99%

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mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the singlecelled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3 ′ ′ ′ ′ ′ -UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.

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Section: Pseudouridylation Of Spliceosomal Rnas Is Tgpus1-independentmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Nakamoto

Lovejoy

Cygan

et al. 2017

RNA

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“…Briefly, the pseudouridylation assay mixture consisting of purified protein (50 nM for RluF and 2 nM for TruA) and in vitro transcript (0.5 M for RluF and 1.5 M for TruA) in the assay buffer (20 mM Tris-HCl, pH 8.0, 0.1 M NH 4 Cl, 2 mM DTT) was incubated at room temperature for 2 h. Following digestion with RNase T1 (150 units) and BAP (0.02 unit) at 37°C for 2 h, the resulting oligonucleotides were extracted with phenol-chloroform, dried in a SpeedVac, and dissolved in sterile water for derivatization with acrylonitrile.…”

Section: Methodsmentioning

confidence: 99%

“…Although the exact mechanism of isomerization is still not clear (4), the incorporation of C5 of the uracil base into the glycosidic bond is catalyzed by either standalone proteins (referred to as ⌿ synthases) (10,11) or RNA-protein complexes (referred to as H/ACA box ribonucleoproteins) (12). Following site-specific recognition, the nitrogen-carbon glycosidic bond is cleaved, and uracil is rotated and then reattached to the sugar, forming a carbon-carbon glycosidic bond on the polyribonucleotide ( Fig.…”

mentioning

confidence: 99%

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

Addepalli

Limbach

2016

Journal of Biological Chemistry

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Pseudouridine is found in almost all cellular ribonucleic acids (RNAs). Of the multiple characteristics attributed to pseudouridine, making messenger RNAs (mRNAs) highly translatable and non-immunogenic is one such feature that directly implicates this modification in protein synthesis. We report the existence of pseudouridine in the anticodon of Escherichia coli tyrosine transfer RNAs (tRNAs) at position 35. Pseudouridine was verified by multiple detection methods, which include pseudouridine-specific chemical derivatization and gas phase dissociation of RNA during liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of total tRNA isolated from E. coli pseudouridine synthase knock-out mutants identified RluF as the enzyme responsible for this modification. Furthermore, the absence of this modification compromises the translational ability of a luciferase reporter gene coding sequence when it is preceded by multiple tyrosine codons. This effect has implications for the translation of mRNAs that are rich in tyrosine codons in bacterial expression systems.Of 150-plus chemical modifications (1, 2) found in cellular RNA, 5-ribosyluridine or pseudouridine (⌿) 3 (3) is the most abundant but still poorly understood (4) modification. Pseudouridine, which has been found in the functionally important regions of RNAs (5), is known to modulate codon-anticodon interactions between mRNA and tRNA (6) and assist assembly of the ribosome (7) and spliceosome (8). The presence of pseudouridine in mRNA makes the message non-immunogenic and highly translatable, thus offering therapeutic opportunities in medical applications (9).Although the exact mechanism of isomerization is still not clear (4), the incorporation of C5 of the uracil base into the glycosidic bond is catalyzed by either standalone proteins (referred to as ⌿ synthases) (10, 11) or RNA-protein complexes (referred to as H/ACA box ribonucleoproteins) (12). Following site-specific recognition, the nitrogen-carbon glycosidic bond is cleaved, and uracil is rotated and then reattached to the sugar, forming a carbon-carbon glycosidic bond on the polyribonucleotide (Fig. 1) by Michael addition-like or glycal mechanisms (13).The ⌿ synthases have been initially classified based on the Escherichia coli enzymes RluA, RsuA, TruA, TruB, and TruD (10, 11). A sixth family with members from archaea and eukaryotes but not from bacteria was added after discovery of Pus10 (14). The enzyme active site carries a catalytically essential aspartate, which is the only absolutely conserved residue in all ⌿ synthases (15, 16). Substrate recognition by the ⌿ synthase is usually in the context of the sequence or structure of the target site in RNA. Site specificity also varies depending on the ⌿ synthase. For example, RsuA isomerizes U516 of 16S rRNA (17) exclusively, whereas the universally conserved ⌿55 of the T⌿C loop in all elongator tRNAs is catalyzed by TruB (18). RluA catalyzes modification of two distinct RNAs, 23S rRNA (position 746) and some tRNAs (position 32) (19). Member...

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RNAModification

Motorin

2015

Encyclopedia of Life Sciences

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Naturally occurring ribonucleic acids (RNAs) contain over 150 chemically altered nucleosides formed by enzymatic modification of the primary RNA transcript during the complex tRNA maturation process. This post‐transcriptional RNA modification is a universally conserved and highly complex metabolic process in the living cell. Cellular RNAs are modified by pure protein stand‐alone enzymes, as well as by s(sno)RNP complexes containing guide s(sno)RNAs. Modified nucleotides present in RNA play an important role in stabilisation of 2D and 3D structures of these molecules, as well as in the fine‐tuning of numerous interactions between RNAs itself and with RNA‐binding protein partners. Modification also protects RNAs against nucleolytic degradation and improves their performance in different interactions in which various RNAs are involved. For example, modifications present in the tRNA anticodon loop are crucially important for correct mRNA decoding during the protein synthesis on the ribosome. Recent progress in the field points out the regulatory character of RNA modification. This emerging concept of ‘RNA‐epigenetics’ supplies the additional level to the regulation of gene expression. This regulation (and deregulation) of RNA‐modification machineries is a basis for some important human pathologies. Key Concepts Cellular RNAs are post‐transcriptionally modified in all life kingdoms RNA modification alters physico‐chemical properties of nucleotides, including their conformation, polarity, hydrophobicity, chemical reactivity and base‐pairing interactions RNA modification is performed by highly specific and regulated enzymatic mechanisms involving pure protein enzymes and catalytic RNA–protein complexes (RNPs) RNA modification is important for regulation of gene expression Transcription‐wide RNA modification is dynamic and regulated cellular process Deregulation of RNA modification may lead to important human pathologies

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Pseudouridine: Still mysterious, but never a fake (uridine)!

Cited by 172 publications

References 212 publications

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

RNAModification

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