Pseudouridine (Ψ, 5-ribosyluracil) is the most abundant RNA modification; in fact, at one time it was considered the "fifth nucleotide". This isomer of uridine is found in various types of RNA, such as snRNA, tRNA, small nucleolar RNA (snoRNA) and many others. Pseudouridine increases the stability of the modified RNA by making the sugar-phosphate backbone more rigid and by facilitating base stacking interactions (pseudouridine contains an extra hydrogen bond donor). When it comes to Watson-Crick base pair interactions, the pseudouridine-adenosine base pair is more stable than the uridine-adenosine base pair; therefore, pseudouridine increases stability. Apart from increasing RNA stability, this modification is also involved in regulation of translation. All eukaryotic stop codons contain one uridine (UAA, UGA and UGA); conversion of this uridine to pseudouridine results in suppression of translational termination and generation of unexpected sense codons. The artificial process of pseudouridylation has an effect on the function of mRNA: it changes the genetic code by making non-canonical base pairing possible in the ribosome decoding center.
Pseudouridylation reactions are catalyzed by enzymes that contain the pseudouridine synthase domain; 13 such enzymes have been identified in humans, which are called pseudouridine syntheses (PUS). These enzymes can be either RNA-dependent or RNA-independent depending on whether a small RNA is needed to guide the enzyme to its target or not. Additionally, different PUS enzymes work in different cell compartments. For instance, PUS4 (also known as TruB pseudouridine synthase family member 1, TRUB1) and PUS7, which are responsible for most of the mRNA pseudouridylation, are located in the nucleus or the cytoplasm. On the other hand, several PUS enzymes, such as PUS1 and TRUB2 are located in the mitochondria, modifying a number of mitochondrial mRNAs (mt-mRNAs). In tRNA, PUS1 and PUS7 modify the second uridine in the UGUAR consensus sequence, as long as this sequence is located in a very structured region of the tRNA.
To date, no pseudouridine erasers or readers have been identified. It is thought that pseudouridylation is most probably an irreversible process.
Pseudouridine is most commonly found in tRNAs, with almost all tRNA molecules having at least one pseudouridine. Therefore, because the addition of pseudouridine happens during the normal processing of tRNA, it is not considered an epitranscriptomic mark. However, pseudouridine acts as an epigenetic mark in mRNAs and ncRNAs of the brain, since pseudouridylation in these two RNAs responds dynamically to stress and differentiation in the cell, giving reason to believe that pseudouridylation may act as an important regulatory mechanism for RNA function. Pseudouridylation in mRNA can be conserved, tissue-specific or inducible, which reflects plasticity and regulatory function. Furthermore, expression of TRBU1, which is mostly expressed in the brain, goes up due to fear conditioning. In addition, expression of the ncRNAs needed to guide RNA-dependent PUS enzymes also goes up in response to fear.