Pseudouridine (abbreviated by the Greek letter psi- Ψ)
is an
isomer
In chemistry, isomers are molecules or polyatomic ions with identical molecular formulae – that is, same number of atoms of each element – but distinct arrangements of atoms in space. Isomerism is existence or possibility of isomers.
Iso ...
of the
nucleoside
Nucleosides are glycosylamines that can be thought of as nucleotides without a phosphate group. A nucleoside consists simply of a nucleobase (also termed a nitrogenous base) and a five-carbon sugar (ribose or 2'-deoxyribose) whereas a nucleotide ...
uridine
Uridine (symbol U or Urd) is a glycosylated pyrimidine analog containing uracil attached to a ribose ring (or more specifically, a ribofuranose) via a β-N1-glycosidic bond. The analog is one of the five standard nucleosides which make up nuclei ...
in which the
uracil
Uracil () (symbol U or Ura) is one of the four nucleobases in the nucleic acid RNA. The others are adenine (A), cytosine (C), and guanine (G). In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by ...
is attached via a carbon-carbon instead of a nitrogen-carbon
glycosidic bond
A glycosidic bond or glycosidic linkage is a type of covalent bond that joins a carbohydrate (sugar) molecule to another group, which may or may not be another carbohydrate.
A glycosidic bond is formed between the hemiacetal or hemiketal group ...
. (In this configuration, uracil is sometimes referred to as 'pseudouracil'.)
Pseudouridine is the most abundant
RNA modification
RNA editing (also RNA modification) is a molecular process through which some cells can make discrete changes to specific nucleotide sequences within an RNA molecule after it has been generated by RNA polymerase. It occurs in all living organisms ...
in cellular
RNA
Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...
. After
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
and following synthesis, RNA can be modified with over 100 chemically distinct modifications. These can potentially regulate RNA expression post-transcriptionally, in addition to the four standard nucleotides and play a variety of roles in the cell including translation, localization and stabilization of RNA. Pseudouridine, being one of them, is the C5-
glycoside
In chemistry, a glycoside is a molecule in which a sugar is bound to another functional group via a glycosidic bond. Glycosides play numerous important roles in living organisms. Many plants store chemicals in the form of inactive glycosides. ...
isomer of uridine that contains a
C-C bond
CC, cc, or C-C may refer to:
Arts, entertainment, and media Fictional characters
* C.C. (''Code Geass''), a character in the ''Code Geass'' anime series, pronounced "C-two"
* C.C. Babcock, a character in the American sitcom ''The Nanny''
* Come ...
between C1 of the
ribose sugar
Ribose is a simple sugar and carbohydrate with molecular formula C5H10O5 and the linear-form composition H−(C=O)−(CHOH)4−H. The naturally-occurring form, , is a component of the ribonucleotides from which RNA is built, and so this compoun ...
and C5 of
uracil
Uracil () (symbol U or Ura) is one of the four nucleobases in the nucleic acid RNA. The others are adenine (A), cytosine (C), and guanine (G). In RNA, uracil binds to adenine via two hydrogen bonds. In DNA, the uracil nucleobase is replaced by ...
, rather than usual C1-N1 bond found in uridine. The C-C bond gives it more rotational freedom and conformational flexibility.
In addition, pseudouridine has an extra hydrogen bond donor at the N1 position.
Also known as 5-ribosyluracil, pseudouridine is a ubiquitous yet enigmatic constituent of structural RNAs (
transfer
Transfer may refer to:
Arts and media
* ''Transfer'' (2010 film), a German science-fiction movie directed by Damir Lukacevic and starring Zana Marjanović
* ''Transfer'' (1966 film), a short film
* ''Transfer'' (journal), in management studies
...
,
ribosomal
Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to fo ...
,
small nuclear (snRNA) and
small nucleolar). Recently it has also been discovered in coding RNA. Being the most abundant, it is found in all 3 phylogenetic domains of life and was the first to be discovered. Pseudouridine accounts for 4% of the nucleotides in the
yeast
Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are estimated to constitut ...
tRNA
Transfer RNA (abbreviated tRNA and formerly referred to as sRNA, for soluble RNA) is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes), that serves as the physical link between the mRNA and the amino ac ...
. This base modification is able to stabilize RNA and improve base-stacking by forming additional hydrogen bonds with water through its extra imino group. There are 11 pseudouridines in the ''
Escherichia coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' rRNA, 30 in yeast cytoplasmic rRNA and a single modification in mitochondrial 21S rRNA and about 100 pseudouridines in human rRNA indicating that the extent of pseudouridylation increases with the complexity of an organism .
Pseudouridine in
rRNA
Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosoma ...
and tRNA has been shown to fine-tune and stabilize the regional structure and help maintain their functions in mRNA decoding, ribosome assembly, processing and translation.
Pseudouridine in
snRNA
Small nuclear RNA (snRNA) is a class of small RNA molecules that are found within the splicing speckles and Cajal bodies of the cell nucleus in eukaryotic cells. The length of an average snRNA is approximately 150 nucleotides. They are transcribed ...
has been shown to enhance
spliceosomal RNA-pre-mRNA interaction to facilitate splicing regulation.
Effects and modification on different RNA
tRNA
![TRNA-Ala yeast](https://upload.wikimedia.org/wikipedia/commons/5/58/TRNA-Ala_yeast.svg)
Ψ is ubiquitous in this class of RNAs and facilitates common
tRNA
Transfer RNA (abbreviated tRNA and formerly referred to as sRNA, for soluble RNA) is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes), that serves as the physical link between the mRNA and the amino ac ...
structural motif
In a polymer, chain-like biological molecule, such as a protein or nucleic acid, a structural motif is a common Biomolecular structure#Tertiary structure, three-dimensional structure that appears in a variety of different, evolutionarily unrel ...
s. One such structural motif is the TΨC stem loop which incorporates Ψ55. Ψ is commonly found in the D stem and anticodon stem and loop of tRNAs from each domain. In each structural motif the unique physicochemical properties of Ψ stabilize structures that would not be possible with the standard U.
During translation Ψ modulates interactions of tRNA molecules with
rRNAs and
mRNAs
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the p ...
. Ψ and other modified nucleotides affect the local structure of the tRNA domains they are found in without impacting the overall fold of the RNA. In the anticodon stem-loop (ASL) Ψ seems critical for proper binding of tRNAs to the ribosome. Ψ stabilizes the dynamic structure of the ASL and promotes stronger binding to the 30S ribosome. The stabilized conformation of the ASL helps maintain correct
anticodon
Transfer RNA (abbreviated tRNA and formerly referred to as sRNA, for soluble RNA) is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes), that serves as the physical link between the mRNA and the amino ac ...
-
codon
The genetic code is the set of rules used by living cells to translate information encoded within genetic material ( DNA or RNA sequences of nucleotide triplets, or codons) into proteins. Translation is accomplished by the ribosome, which links ...
pairings during translation. This stability may increase translational accuracy by decreasing the rate of peptide bond formation and allowing for more time for incorrect codon-anticodon pairs to be rejected. Despite Ψ’s role in local structure stabilization, pseudouridylation of tRNA is not essential for cell viability and is not usually required for
aminoacylation
Aminoacylation is the process of adding an aminoacyl group to a compound.
See also
*Acylation
* tRNA aminoacylation
*Transfer RNA-like structures
Transfer RNA-like structures (tRNA-like structures) are RNA sequences, which have a similar tertiar ...
.
mRNA
Ψ is also found in
mRNAs
In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein.
mRNA is created during the p ...
which are the template for protein synthesis. Ψ residues in mRNA can affect the coding specificity of stop codons UAA, UGA, and UAG. In these stop codons both a U→Ψ modification and a U→C mutation both promote nonsense suppression. In the SARS-CoV2 vaccine from BioNTech/Pfizer, also known as
BNT162b2, Tozinameran or Comirnaty, all U's have been substituted with
N1-methylpseudouridine, a nucleoside related to Ψ that contains a methyl group added to N1 atom.
rRNA
Ψ is found in the large and small
ribosomal
Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to fo ...
subunits of all domains of life and their
organelle
In cell biology, an organelle is a specialized subunit, usually within a cell, that has a specific function. The name ''organelle'' comes from the idea that these structures are parts of cells, as organs are to the body, hence ''organelle,'' the ...
s. In the ribosome Ψ residues cluster in domains II, IV, and V and stabilize RNA-RNA and/or RNA-protein interactions. The stability afforded by Ψ may assist
rRNA
Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosoma ...
folding and ribosome assembly. Ψ may also influence the stability of local structures which impact the speed and accuracy of decoding and proofreading during translation.
snRNA
Ψ is found in the major
spliceosomal snRNAs of eukaryotes. Ψ residues in snRNA are often phylogenetically conserved, but have some variations across taxa and organisms. The Ψ residues in snRNAs are normally located in regions that participate in RNA-RNA and/or RNA-protein interactions involved in the assembly and function of the spliceosome. Ψ residues in snRNAS contribute to the proper folding and assembly of the spliceosome which is essential for pre-mRNA processing.
Pseudouridine synthase proteins
Pseudouridine are RNA modifications that are done
post-transcription, so after the RNA is formed . The proteins that do this modification are called Pseudouridine Synthases or PUS. PUS proteins are found in all kingdoms of life. Most research has been done on how PUS proteins modify tRNA, so mechanisms involving snRNA, and mRNA are not clearly defined. PUS proteins can vary on RNA specificity, structure, and
isomerization
In chemistry, isomerization or isomerisation is the process in which a molecule, polyatomic ion or molecular fragment is transformed into an isomer with a different chemical structure. Enolization is an example of isomerization, as is tautomeriz ...
mechanisms. The different structures of PUS are divided into 5 families. The families share the active sequence and important structural motifs.
TruA
TruA domain modifies a variety of different places in tRNA, snRNA, and mRNA. The mechanism of isomerization of uridine is still being talked about in this family.
![PDB 2nr0 EBI](https://upload.wikimedia.org/wikipedia/commons/5/51/PDB_2nr0_EBI.jpg)
PUS 1 is located in the nucleus and modifies tRNA at different locations, U44 of U2 snRNA, and U28 of U6 snRNA. Studies found that PUS 1 expression increased during environmental stress and is important for regulating the splicing of RNA. Also, that PUS 1 is necessary for taking the tRNA made in the nucleus and sending them to the cytoplasm.
PUS 2 is very similar to PUS 1, but located in the mitochondria and only modifies U27 and U28 of mito-tRNA. This protein modifies the mitochondrial tRNA, which has a lesser amount of pseudouridine modifications compared to other tRNAs. Unlike most mitochondria located protein, PUS 2 has not been found to have a mitochondrial targeting signal or MTS.
PUS 3 is a homolog to PUS 1, but modifies different places of the tRNA (U38/39) in the cytoplasm and mitochondrial. This protein is the most conserved of the TruA family. A decrease in modifications made by PUS 3 was found when the tRNA structure of improperly folded. Along with tRNA the protein targets ncRNA and mRNA, further research is still needed as to the importance of this modification. PUS 3 along with PUS 1 modify the steroid activator receptor in humans.
TruB
The TruB family only contains PUS 4 located in the mitochondrial and nucleus. PUS 4 modification is heavily conserved located in the U55 in the elbow of the tRNA. The human form of PUS 4 is actually missing a binding domain called PUA or pseudouridine synthase and
archaeosine trans-glycosylase. PUS 4 has a sequence specificity for T-loop part of the tRNA. Preliminary data of PUS4 modifying mRNA, but more research is needed to confirm. Also binds to a specific Brome Mosaic Virus, which is a plant-infecting RNA virus.
TruD
TruD is able to modify a variety of RNA, and it is unclear how these different RNA substrates are recognized. PUS 7 modifies U2 snRNA at the position 35 and this modification will increase when the cells are in heat shock. Another modification is cytoplasmic tRNA in position 13, and position 35 in pre-tRNA
Tyr. PUS 7 modifies almost specificity does not depend on the type of RNA as mRNA show pseudouridylated by PUS 7. Recognize this the sequence of the RNA, UGUAR with the second U being the nucleotide that will be modified. The pseudouridylation of mRNA by PUS 7 increases during heat shock, because the protein moves from the nucleus to the cytoplasm. The modification is thought to increase the stability of mRNA during heat shock before the RNA goes to the nucleus or mitochondria, but more studies are needed.
RluA
The RluA domain of these proteins can identify the substrate through a different protein binding to the substrate and then particular bonds to the RluA domain.
PUS 5 is not well studied and located pseudouridine synthase and similar to Pus 2 does not have a mitochondrial signal targeting sequence. The protein modifies U2819 of mitochondrial 21S rRNA. Also suspected that Pus 5 modifies some Uridines in the mRNA, but again more data is needed to confirm.
PUS 6 has one that only modifies U31 of cytoplasmic and mitochondrial tRNA. Pus 6 is also known to modify mRNA.
PUS 8 also known as Rib2 modifies cytoplasmic tRNA at position U32. On the C-terminus there is a DRAP-deaminase domain related to the biosynthesis of riboflavin. The RluA and DRAP or deaminase domain related to
riboflavin synthase
Riboflavin synthase is an enzyme that catalyzes the final reaction of riboflavin biosynthesis. It catalyzes the transfer of a four-carbon unit from one molecule of 6,7-dimethyl-8-ribityllumazine onto another, resulting in the synthesis of ribof ...
have completely separate functions in the protein and it is not known whether they interact with each other. PUS 8 is necessary in yeast, but that is suspected to be related to the riboflavin synthesis and not the pseudouridine modification.
PUS 9 and PUS 8 catalyze the same position in mitochondrial tRNA instead of cytoplasmic. It is the only PUS protein that contains a mitochondrial targeting signal domain on the N-terminus. Studies suspect that PUS 9 can modify mRNAs, which would mean less substrate specificity.
RsuA
Techniques in genome sequencing for pseudouridine
Pseudouridine can be identified through a multitude of different techniques. A common technique to identify modifications in RNA and DNA is Liquid Chromatography with Mass Spectrometry or
LC-MS.
Mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
separates molecules by the mass and charge. While uridine and pseudouridine have the same mass, but have different charges.
Liquid chromatography
In chemical analysis, chromatography is a laboratory technique for the Separation process, separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it ...
works by retention time, which has to do with leaving the column. A chemical way to identify pseudouridine uses a compound called CMC or N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide to specifically label and distinguish Uridine from Pseudouridine. CMC will bond both with Pseudouridine and Uridine, but holds tighter to the former, because of the third nitrogen able to form hydrogen bond. CMC bound to pseudouridine can then be imaged by tagging a signaling molecule. This method is still being worked on to become high-throughput.
Medical relevance of pseudouridine
Pseudouridine exerts a subtle but significant influence on the nearby sugar-phosphate backbone and also enhances base stacking. These effects may underlie the biological role of most, but perhaps not all of the pseudouridine residues in RNA. Certain genetic mutants lacking specific pseudouridine residues in tRNA or rRNA exhibit difficulties in translation, display slow growth rates, and fail to compete effectively with wild-type strains in mixed culture. Pseudouridine modifications are also implicated in human diseases such as
mitochondrial myopathy
Mitochondrial myopathies are types of myopathies associated with mitochondrial disease. On biopsy, the muscle tissue of patients with these diseases usually demonstrate "ragged red" muscle fibers. These ragged-red fibers contain mild accumulation ...
and
sideroblastic anemia
Sideroblastic anemia, or sideroachrestic anemia, is a form of anemia in which the bone marrow produces ringed sideroblasts rather than healthy red blood cells (erythrocytes). In sideroblastic anemia, the body has iron available but cannot incorpora ...
(MLASA) and Dyskeratosis congenita.
Dyskeratosis congenita
Dyskeratosis congenita (DKC), also known as Zinsser-Engman-Cole syndrome, is a rare progressive congenital disorder with a highly variable phenotype. The entity was classically defined by the triad of abnormal skin pigmentation, nail dystrophy, and ...
and
Hoyeraal-Hreidarsson syndrome are two rare inherited syndromes caused by mutations in
DKC1
H/ACA ribonucleoprotein complex subunit 4 is a protein that in humans is encoded by the gene ''DKC1''.
This gene is a member of the snoRNA, H/ACA snoRNPs (small nucleolar ribonucleoproteins) gene family. snoRNPs are involved in various aspects of ...
, the gene encoding for the pseudouridine synthase dyskerin. Pseudouridines have been recognized as regulators of viral latency processes in human immunodeficiency virus (
HIV
The human immunodeficiency viruses (HIV) are two species of ''Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immune ...
) infections.
Pseudouridylation has also been associated with the pathogenesis of maternally inherited diabetes and deafness (MIDD). In particular, a point mutation in a mitochondrial tRNA seems to prevent the pseudouridylation of one nucleotide, thus altering the tRNA tertiary structure. This may lead to higher tRNA instability, causing deficiencies in mitochondrial translation and respiration.
Vaccines
When pseudouridine is used in place of uridine in synthetic mRNA, the modified mRNA molecule arouses less response from
Toll-like receptors
Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single-pass membrane-spanning receptors usually expressed on sentinel cells such as macrophages and dendritic cells, that recognize st ...
, a part of the human immune system that would otherwise identify the mRNA as unwelcome. This makes pseudouridine useful in
mRNA vaccines
An mRNA vaccine is a type of vaccine that uses a copy of a molecule called messenger RNA (mRNA) to produce an immune response. The vaccine RNA transfection, delivers molecules of antigen-encoding mRNA into dendritic cell, immune cells, which us ...
, including the mRNA
COVID-19 vaccine
A COVID19 vaccine is a vaccine intended to provide acquired immunity against severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2), the virus that causes coronavirus disease 2019 ( COVID19).
Prior to the COVID19 pandemic, an e ...
s. This property of pseudouridine was discovered by
Katalin Karikó
Katalin Karikó ( hu, Karikó Katalin, ; born 17 January 1955) is a Hungarian-American biochemist who specializes in RNA-mediated mechanisms. Her research has been the development of in vitro- transcribed mRNA for protein therapies. She co-fo ...
and
Drew Weissman
Drew Weissman (born 1959) is an American physician-scientist best known for his contributions to RNA biology. His work helped enable development of effective mRNA vaccines, the best known of which are those for COVID-19 produced by BioNTech/P ...
in 2005.
N1-Methylpseudouridine provides even less innate immune response than Ψ, as well as improving
translation
Translation is the communication of the Meaning (linguistic), meaning of a #Source and target languages, source-language text by means of an Dynamic and formal equivalence, equivalent #Source and target languages, target-language text. The ...
capacity.
Both Pfizer-BioNTech and Moderna mRNA vaccines therefore use N1-Methylpseudouridine rather than Ψ.
See also
*
Pseudouridine kinase
In enzymology, a pseudouridine kinase () is an enzyme that catalyzes the chemical reaction
:ATP + pseudouridine \rightleftharpoons ADP + pseudouridine 5'-phosphate
Thus, the two substrates of this enzyme are ATP and pseudouridine, whereas its ...
*
TRNA-pseudouridine synthase
*
PUS1
References
{{reflist
Nucleosides
Pyrimidinediones