Post-transcriptional pseudouridylation in mRNA as well as in some major types of noncoding RNAs

Abstract

Pseudouridylation is a post-transcriptional isomerization reaction that converts a uridine to a pseudouridine (Ψ) within an RNA chain. Ψ has chemical properties that are distinct from that of uridine and any other known nucleotides. Experimental data accumulated thus far have indicated that Ψ is present in many different types of RNAs, including coding and noncoding RNAs. Ψ is particularly concentrated in rRNA and spliceosomal snRNAs, and plays an important role in protein translation and pre-mRNA splicing, respectively. Ψ has also been found in mRNA, but its function there remains essentially unknown. In this review, we discuss the mechanisms and functions of RNA pseudouridylation, focusing on rRNA, snRNA and mRNA. We also discuss the methods, which have been developed to detect Ψs in RNAs. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

Figure 1.

Pseudouridine properties. (A) U-to-Ψ isomerization. Uridine (U) and pseudouridine (Ψ) are indicated. N1 (red) of the base and the N3-C6 axis are also indicated, a: hydrogen bond acceptor; d: hydrogen bond donor. (B) Water-mediated hydrogen-bond network. Depicted is the Ψ-specific, water-mediated hydrogen-bond network. The water molecule is indicated. The positions of the base and the sugar are also indicated. (C) Sugar ring C3’-endo configuration. Shown is the C3’-endo configuration of the sugar ring, established upon the formation of a Ψ-specific, water-mediated hydrogen bond network. Carbon numbers are indicated.

Figure 2.

Pseudouridylation assays. (A) Assay based on CMC-modification followed by primer-extension. The carbodiimide CMC is able to covalently attach to G, U and Ψ. Upon alkaline treatment, G-CMC and U-CMC adducts are reversed, but CMC remains covalently bound to Ψ (Ψ-CMC). Subsequent primer-extension results in stops one nucleotide before the Ψ-CMC adducts. CMC-nucleotide adducts are indicated. Primer (red lines)-extension (dotted red lines) stops are indicated as well. * represents the radio label. (B) Assay based on site-specific cleavage/labeling, nuclease digestion and TLC. An RNA is site-specifically cleaved by RNase H directed by a complementary 2’-O-methyl RNA-DNA chimera (here, cleavage is directed at the site 3’ of the U/Ψ site). The 5’ cleaved fragment is ligated to a known, 5’ labeled RNA oligonucleotide. After gel purification, the ligated, radiolabeled RNA is digested with T2. The digestion products Ψ³²p and U³²p are separated on TLC. The chimeric oligonucleotide (4 2’-deoxy nucleotides flanked by 2’-O-methylated nucleotides) is depicted (green). The cleavage site is indicated by a small arrow. The bridging deoxyoligonucleotide (blue) and known 5’ labeled RNA oligonucleotide are also shown. On TLC, the Ψ³²ρ and U³²p spots are radiolabeled and visible; the Ap, Gp and Cp are not labeled and not visible.

Figure 3.

Box H/ACA guided RNA pseudouirdylation. The components of a box H/ACA RNP, including one box H/ACA RNA (black line with box H and box ACA) and four core proteins (Nhp2, Nop10, Gar1 and Cbf5), are shown. The red line represents the substrate RNA. N represents any nucleotide; Ψ (indicated by an arrow) is the modified nucleotide.

Figure 4.

Nonsense suppression induced by targeted pseudouridylation. Gene expression (from DNA to RNA to protein) is schematized. Black letters represent wild-type sequences; red letters and red arrows indicate mutations (present in disease genes) and the consequences of mutations, respectively; the blue arrow indicates U-to-Ψ conversion; the green letter and green lines/arrow depict the isomerized nucleotide (Ψ) and the consequences, respectively.

Figure 5.

Ψs in gene expression. Pseudouridylated coding RNAs (pre-mRNA and mRNA) and non-coding RNAs (snRNA, tRNA and rRNA) are shown in red. Ψs of snRNA contribute to pre-mRNA splicing; Ψs of tRNA aid in RNA stability and translation fidelity; Ψs of rRNA are involved in ribosome biogenesis and translation. Although pseudouridylation of stop codons promotes stop codon read-through and suppresses NMD, the function of Ψs in other sites/regions of mRNA is not clear. The blue question marks and keywords indicate speculative functions.