Pseudouridine is an isomer of uridine. It is the most abundant modified nucleoside found in non-coding RNAs (tRNA, rRNA, snRNA) and is also present in mRNA. It functions by stabilizing RNA structure through enhanced base stacking and increased hydrogen bonding capabilities. Artificial pseudouridine incorporation into mRNA can change the genetic code by facilitating non-canonical base pairing in the ribosome decoding center [1].

Pseudouridine in RNA can be specifically modified with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) under denaturing conditions. This modification creates a block to reverse transcriptase (RT), causing it to stop one nucleotide 3' to the pseudouridylated site. This property is the basis for its biochemical detection [1].

The mechanism of box H/ACA RNP-catalyzed pseudouridylation has been elucidated through structural studies. Crystal structures of archaeal box H/ACA RNPs show that the Cbf5 protein (the pseudouridylase) interacts with the box H/ACA RNA and the substrate RNA. The substrate RNA base-pairs with the guide sequence in the pseudouridylation pocket, positioning the target uridine precisely at the Cbf5 catalytic site for modification. The proteins Nop10 and L7Ae (Nhp2 in eukaryotes) interact with the upper stem of the RNA, while Gar1 binds only to Cbf5 and is involved in product release [3].
NMR solution structures of guide-substrate complexes indicate an unusual base-pairing topology: the substrate RNA base-pairs with the guide sequence from one side only, resulting in a U-shaped or Ω-shaped substrate structure. This mode of interaction allows successive loading and release of substrate RNAs [3].
Reconstitution systems for box H/ACA RNP activity have identified three important sequence and structural elements: the stability of the hairpin structure harboring the guide sequence (the pseudouridylation pocket), the stability of base-pairing between the guide sequence and target RNA, and the distance (obligatory 14-16 nt) between the target uridine and box H or ACA [3].

The Pseudo-seq method was developed to identify pseudouridine sites in the transcriptome. The detailed experimental procedure is as follows:
Total RNA was isolated from yeast or HeLa cells. PolyA+ RNA was isolated from total RNA using oligo dT cellulose beads. For some libraries, two sequential rounds of polyA selection were performed. RNA was fragmented in ZnCl₂ or ZnAcetate solution under specific time and temperature conditions. Fragmented RNA was then precipitated. For CMC treatment, RNA was denatured in EDTA at 80°C and then placed on ice. CMC in BEU buffer (7 M urea, EDTA, bicine, pH 8.5) was added to a final concentration of 0.2 or 0.4 M. CMC modification was carried out at 40°C for 30 min, followed by ethanol precipitation. Subsequent reversal of modification of Us and Gs was carried out in sodium-carbonate buffer (pH 10.4, EDTA) at 50°C for 2 hours, followed by precipitation. Mock-treated samples were incubated in BEU buffer without CMC and processed in parallel. RNA fragments were dephosphorylated, and fragments of 80-140 nt were size-selected on a urea-TBE PAGE gel. A pre-adenylated 3' adaptor was ligated to the RNA fragments using T4 RNA ligase. Reverse transcription was carried out using AMV-RT. Truncated cDNAs, which stop one nucleotide before the pseudouridine site, were size-selected and purified on a urea-TBE PAGE gel. cDNAs were circularized with circLigase and amplified by PCR. The final libraries were sequenced on an Illumina HiSeq 2000 [1].
Using Pseudo-seq, pseudouridine sites were identified in mRNAs of both yeast and human cells. In yeast grown to high density, 260 pseudouridine sites were identified in 238 protein-coding transcripts. In human HeLa cells, 96 pseudouridine sites were identified in 89 mRNAs. These modifications were found in 5' transcript leaders, coding sequences, and 3' untranslated regions. The process was found to be regulated by environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells [1].
In yeast, genetic analysis using deletion strains of pseudouridine synthase (PUS) genes allowed assignment of many modification sites to specific enzymes. For example, mRNA targets were identified for Pus1, Pus2, Pus3, Pus4, Pus6, Pus7, and Pus9. Pus4 and Pus7 targets contained clear sequence consensus sites (GUUCNANNC for Pus4; UGUAR for Pus7) [1].
In yeast, pseudouridine sites were also identified in non-coding RNAs. 74 novel pseudouridylated sites were found in ncRNAs, including in snoRNAs (e.g., snR37, snR43, snR49, snR81), RNase MRP RNA (NME1), and U5 snRNA. Many of these were also regulated by growth conditions [1].
In human HeLa cells, novel pseudouridine sites were discovered in ncRNAs, including 4 previously unknown sites in rRNA, as well as sites in lncRNAs (e.g., MALAT1), snRNAs, and snoRNAs (e.g., RN7SK) [1].

This study did not involve the administration of pseudouridine as a drug to animals. It used yeast strains and cultured human HeLa cells. Yeast strains (wild-type and various pus and snoRNA deletion mutants) were grown in YPAD medium at 30°C and harvested by centrifugation either in log phase (OD ~1) or at high density (OD ~12-15). HeLa cells were cultured in DMEM with 10% FBS at 37°C with 5% CO₂. For serum starvation experiments, cells were plated, washed, and then incubated in either serum-free medium or full medium for 24 hours [1].

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This study did not involve the administration of pseudouridine as a drug to animals. It used yeast strains and cultured human HeLa cells. Yeast strains (wild-type and various pus and snoRNA deletion mutants) were grown in YPAD medium at 30°C and harvested by centrifugation either in log phase (OD ~1) or at high density (OD ~12-15). HeLa cells were cultured in DMEM with 10% FBS at 37°C with 5% CO₂. For serum starvation experiments, cells were plated, washed, and then incubated in either serum-free medium or full medium for 24 hours [1].

[1]. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature. 2014 Nov 6;515(7525):143-6.

[2]. Pseudouridine in RNA: what, where, how, and why. IUBMB Life. 2000 May;49(5):341-51.

[3]. RNA pseudouridylation: new insights into an old modification. Trends Biochem Sci. 2013 Apr;38(4):210-8.

[4]. Eukaryotic stand-alone pseudouridine synthases - RNA modifying enzymes and emerging regulators of gene expression? RNA Biol. 2017 Sep 2;14(9):1185-1196.

Pseudouridine is a C-glycopyrimidine compound composed of uracil linked to a β-D-furanose ribose residue at the 5-position. It is the C-glycosyl isomer of the nucleoside uridine and is an important metabolite. Pseudouridine is a metabolite found or produced in Escherichia coli (strains K12 and MG1655). It has been reported in Drosophila melanogaster, Mycoplasma gallisepticum, and other organisms with relevant data. β-Pseudouridine is a metabolite found or produced in Saccharomyces cerevisiae. It is a naturally occurring uridine isomer in RNA, where the ribosome is attached to a carbon atom instead of a nitrogen atom.

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Pseudouridine is the most abundant modified nucleoside in non-coding RNAs. Its presence enhances the function of tRNA and rRNA by stabilizing RNA structure. While mRNAs were not previously known to contain pseudouridine, this study (Pseudo-seq) demonstrates that endogenous mRNAs are specifically pseudouridylated in a highly regulated manner in both yeast and human cells [1].
Artificial pseudouridylation of mRNA has been shown to dramatically affect its function by changing the genetic code, as it facilitates non-canonical base pairing in the ribosome decoding center [1].
The regulated nature of mRNA pseudouridylation suggests a mechanism for the rapid and regulated rewiring of the genetic code in response to environmental signals [1].
Mutations in pseudouridine synthases (PUS genes) are associated with human diseases, including mitochondrial myopathy and sideroblastic anemia (MLASA, linked to PUS1 mutations), dyskeratosis congenita (linked to Dyskerin, the human homolog of yeast Cbf5), and lung cancer (linked to snoRNA 42). This work suggests these diseases could be due to misregulation of mRNA targets [1].

C9H12N2O6

244.203

244.069

1445-07-4

15047

White to off-white solid powder

1.7±0.1 g/cm3

598.4±60.0 °C at 760 mmHg

231 °C(dec.)

315.7±32.9 °C

0.0±1.8 mmHg at 25°C

1.697

-3.77

5

6

2

17

382

4

O1[C@]([H])(C([H])([H])O[H])[C@]([H])([C@]([H])([C@]1([H])C1=C([H])N([H])C(N([H])C1=O)=O)O[H])O[H]

PTJWIQPHWPFNBW-GBNDHIKLSA-N

InChI=1S/C9H12N2O6/c12-2-4-5(13)6(14)7(17-4)3-1-10-9(16)11-8(3)15/h1,4-7,12-14H,2H2,(H2,10,11,15,16)/t4-,5-,6-,7+/m1/s1

5-[(2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-pyrimidine-2,4-dione

Pseudouridine NSC-162405 NSC162405NSC 162405

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~511.88 mM)
H2O : ~25 mg/mL (~102.38 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 25 mg/mL (102.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)