Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: A new approach with future therapeutic potential

Boros, Gábor; Mikó, Edit; Muramatsu, Hiromi; Weissman, Drew; Emri, Eszter; Rózsa, Dávid; Nagy, Gábor; Juhász, Attila; Juhász, István; Horst, Gijsbertus T. J. van der; Horkay, Irén; Remenyik, Éva; Karikó, Katalin; Emri, Gabriella

doi:10.1016/j.jphotobiol.2013.09.010

Cited by 28 publications

(33 citation statements)

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“…Such interactions seem to play a crucial role in amino acylation fidelity and efficiency in yeast (52) where ⌿ interactions with adenosine strengthen the anticodon-codon/stop codon interactions (51,53,54). These findings might explain why incorporation of pseudouridine into mRNA makes it highly stable and translatable, offering a number of opportunities for medical applications (9).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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“…A Potorous tridactylus fotoliáz génjét kódoló mRNS-t kollaboráció keretében Karikó Katalin laboratóriuma in vitrotranszkripcióval hozta létre. A szekvencia kodon-optimalizálásával, az mRNS pszeudouridin-módosításával, hosszú poly(A) farok és cap1 struktúra kialakításával, valamint az mRNS HPLC tisztításával összességében magas transzlációs hatékonyságot, és biológiai stabilitást sikerült elérni, amit a DE OEC Bôrgyógyászati Klinika kutatócsoportja igazolt (34). Kísérleteink elsô fázisában kimutattuk, hogy a transzfektált, majd fotoreaktivált humán keratinocitákban a CPD-k száma több mint 60%-kal csökkent a fotoliáz aktivációjához szükséges energiaforrást nélkülözô sejtekhez képest, ami bizonyította, hogy a transzlált fehérje funkcionálisan is aktív.…”

Section: Nukleozid-módosított Fotoliáz Mrns In Vitro Vizsgálatai éS Aunclassified

“…Kísérleteink elsô fázisában kimutattuk, hogy a transzfektált, majd fotoreaktivált humán keratinocitákban a CPD-k száma több mint 60%-kal csökkent a fotoliáz aktivációjához szükséges energiaforrást nélkülözô sejtekhez képest, ami bizonyította, hogy a transzlált fehérje funkcionálisan is aktív. A fotoreaktivá-ció szignifikánsan csökkentette az UVB dózis antiproliferatív hatását (34). A fotoliáz mRNS keratinocitákba juttatása alkalmasnak tûnik az UVB-indukálta CPD léziók eltávolítására humán keratinocitákban, ami elôrevetíti alkalmazási lehetôségét a bôrdaganatok megelôzésében.…”

Section: Nukleozid-módosított Fotoliáz Mrns In Vitro Vizsgálatai éS Aunclassified

Az mRNS-alapú génterápia dermatológiai alkalmazásának lehetôségei: fényvédelem újragondolva

Boros¹,

Mikó²,

Horkay³

et al. 2013

BVSZ

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“…15,41 A prominent example is a C residue in eukaryotic U2 RNA that stabilizes and fine-tunes spliceosomal branchsite interaction 39,173 , involving a water-CNH1 hydrogen bond. 174,175 Functional importance for RNA C in artificial mRNAs Synthetic replacement of all uridines by C renders mRNAs non-immunogenic 176 , increases biological stability [176][177][178] and enhances translation in vivo 176,179,180 , while reducing PKR activation. 181 In contrast, studies with in vitro assays suggested that mRNAs where all Us were changed to Cs inhibit translation at the initiation and elongation levels.…”

mentioning

confidence: 99%

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

Spenkuch

Motorin

Helm³

2014

RNA Biology

179

186

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Pseudouridine (C) is the most abundant of >150 nucleoside modifications in RNA. Although C was discovered as the first modified nucleoside more than half a century ago, neither the enzymatic mechanism of its formation, nor the function of this modification are fully elucidated. We present the consistent picture of C synthases, their substrates and their substrate positions in model organisms of all domains of life as it has emerged to date and point out the challenges that remain concerning higher eukaryotes and the elucidation of the enzymatic mechanism.

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Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: A new approach with future therapeutic potential

Cited by 28 publications

References 58 publications

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

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

Az mRNS-alapú génterápia dermatológiai alkalmazásának lehetôségei: fényvédelem újragondolva

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

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