RNA pseudouridylation: new insights into an old modification

Abstract

Pseudouridine is the most abundant post-transcriptionally modified nucleotide in various stable RNAs of all organisms. Pseudouridine is derived from uridine via base-specific isomerization, resulting in an extra hydrogen-bond donor that distinguishes it from other nucleotides. In eukaryotes, uridine-to-pseudouridine isomerization is catalyzed primarily by box H/ACA RNPs, ribonucleoproteins that act as pseudouridylases. When introduced into RNA, pseudouridine contributes significantly to RNA-mediated cellular processes. It was recently discovered that pseudouridylation can be induced by stress, suggesting a regulatory role for pseudouridine. It has also been reported that pseudouridine can be artificially introduced into mRNA by box H/ACA RNPs and that such introduction can mediate nonsense-to-sense codon conversion, thus demonstrating a new means of generating coding or protein diversity.

Fig. 1

Schematic representation of U-to-Ψ isomerization. The structures of uridine (U) and Ψ are shown. Ψ is derived from U through the isomerization reaction where the base is rotated 180° along the N3-C6 axis and the C5-C1′ bond forms. The nitrogen at position 1 in U and the extra hydrogen bond donor in Ψ are indicated (red). Hydrogen bond acceptor (a) and hydrogen bond donor (d) are also indicated.

Fig. 2

Schematic depiction of eukaryotic box H/ACA RNP-catalyzed pseudouridylation. Box H/ACA RNP, consisting of one guide RNA with a hairpin-hinge-hairpin-tail-structure (black line) and four core proteins, Cbf5, Nhp2, Nop10 and Gar1 (color-coded ovals), is shown. The substrate RNA (red line), which is paired with the guide sequences in the pseudouridylation pockets of box H/ACA RNA, is also shown. Ψ (red) is the target nucleotide converted from uridine, and N (red) represents any nucleotide. Boxes H and ACA of the guide RNA are indicated. Although box H/ACA RNA is usually a double-hairpin molecule in eukaryotic cells, it appears that the two hairpins function independently (each is an independent functional unit).

Fig. 3

Ψs are clustered in functionally important regions of rRNAs and snRNAs. The secondary structures of yeast 18S and 25S–5.8S rRNAs (A) and the primary sequences and secondary structures of vertebrate snRNAs (B) are depicted. The red squares represent Ψs. The peptidyl transferase center (PTC), the A-site finger (ASF), and Helix 69 of 25S rRNA are indicated. Some important regions of snRNAs, including the 5′ end of U1, the branch site-recognition region of U2, and the loop sequence of U5, are indicated by thick lines. The red arrows mark the Ψs (in U1 and U2) that are conserved across species (including yeast). The blue arrows indicate the Ψs (in U2) that can be induced by stress. The 5′-cap structures of snRNAs are also shown.

Fig. 3

Ψs are clustered in functionally important regions of rRNAs and snRNAs. The secondary structures of yeast 18S and 25S–5.8S rRNAs (A) and the primary sequences and secondary structures of vertebrate snRNAs (B) are depicted. The red squares represent Ψs. The peptidyl transferase center (PTC), the A-site finger (ASF), and Helix 69 of 25S rRNA are indicated. Some important regions of snRNAs, including the 5′ end of U1, the branch site-recognition region of U2, and the loop sequence of U5, are indicated by thick lines. The red arrows mark the Ψs (in U1 and U2) that are conserved across species (including yeast). The blue arrows indicate the Ψs (in U2) that can be induced by stress. The 5′-cap structures of snRNAs are also shown.

Fig. 4

Pseudouridylation can be induced by stress. As indicated, snR81, a yeast box H/ACA RNA, normally uses its 5′ pseudouridylation pocket to direct the formation of Ψ42 (red arrow) in U2, and its 3′ pseudouridylation pocket to guide the formation of Ψ1051 (red arrow) in 25S rRNA. Under normal conditions, the 3′ pseudouridylation pocket cannot direct the conversion of U93 (black arrow) into Ψ93 in U2 snRNA. However, under stress conditions, the 3′ pseudouridylation pocket becomes capable of directing the formation of Ψ93 (green arrow), despite the fact that there are two U-U mismatches (indicated) between the 3′ pseudouridylation pocket and the U2 sequence flanking position 93. The sequence of snR81 and partial sequences of U2 and 25S rRNA are shown. Boxes H and ACA within snR81 are indicated.

Fig. 5

Schematic depiction of Ψ-mediated nonsense suppression. When the invariant uridine of a stop codon is converted into Ψ, the modified stop codon ΨAA in the ribosomal A-site is no longer recognized by release factors (RF). Instead, ΨAA is recognized by a specific aminoacylated tRNA (depicted as EF-Tu/GTP/aa-tRNA ternary complex), allowing the incorporation of a specific amino acid into the elongating peptide and resulting in nonsense suppression. The ribosome and the ribosomal A-, P- and E-sites are indicated. tRNAs, mRNA and elongating peptide are also depicted. The small solid blue circles represent amino acids.

Fig. 6

mRNA pseudouridylation as a novel means to produce protein diversity. The pathway of gene expression from DNA (blue) to mature mRNA (red) to protein is depicted. Each codon consists of three nucleotides and is indicated by an underline. When mRNA is pseudouridylated, the modified mRNA (purple) may encode a protein different from that encoded by the un-modified mRNA (red). The question mark indicates the possible differences between the two proteins.