Transcriptome-wide dynamics of RNA pseudouridylation

Abstract

Pseudouridylation is the most abundant internal post-transcriptional modification of stable RNAs, with fundamental roles in the biogenesis and function of spliceosomal small nuclear RNAs (snRNAs) and ribosomal RNAs (rRNAs). Recently, the first transcriptome-wide maps of RNA pseudouridylation were published, greatly expanding the catalogue of known pseudouridylated RNAs. These data have further implicated RNA pseudouridylation in the cellular stress response and, moreover, have established that mRNAs are also targets of pseudouridine synthases, potentially representing a novel mechanism for expanding the complexity of the cellular proteome.

Figure 1. Constitutive and inducible pseudouridylation

a | Schematic of constitutive uridine-to-pseudouridine (ψ) conversions catalysed by box H/ACA ribonucleoproteins (RNPs) or by a stand-alone protein, pseudouridine synthase 7 (Pus7). Box H/ACA RNPs (top) consist of a small box H/ACA RNA (which has a typical hairpin–hinge–hairpin– tail structure) and four core proteins, namely centromere-binding factor 5 (Cbf5; known as dyskerin in mammals), glycine-arginine-rich protein 1 (Gar1), non-histone protein 2 (Nhp2) and nucleolar protein 10 (Nop10). Cbf5 is the PUS. The substrate RNA engages the box H/ACA RNP via complementary base-pair interactions with the pseudouridylation pocket (thick lines) of the box H/ACA RNA. The uridine targeted for modification and its 3′ adjacent nucleotide (N) are positioned at the base of the upper stem and remain unpaired throughout the reaction. Pus7 (bottom), a stand-alone PUS, recognizes and catalyses pseudouridylation of its substrate (the U2 small nuclear RNA (snRNA) substrate sequence is shown as an example). b | Schematic of a stress-induced uridine-to-pseudouridine conversion catalysed by a box H/ACA RNP or by Pus7. The 3′ hairpin complex (containing the 3′ pseudouridylation pocket) of a box H/ACA RNP is shown (top) with two mismatches between the guide sequence and its substrate (red crosses). Induced pseudouridylation of U2 by Pus7 is also shown (bottom). The nucleotides that differ from the constitutive Pus7 recognition sequence shown in part a are highlighted in blue.

Figure 2. Possible roles of pseudouridines in gene regulation

Pseudouridine (Ψ) nucleosides are introduced into pre-mRNAs at coding and non-coding exons and presumably also at introns. Given that they are present in pre-mRNAs and mRNAs, as well as in non-coding RNAs (such as spliceosomal small nuclear RNAs (snRNAs), ribosomal RNAs (rRNAs) and tRNAs) that are involved in every step in the pathway (splicing, translation and mRNA decay), pseudouridylation is likely to have a complex role in the regulation of gene expression. In fact, the functions of some pseudouridine residues in snRNAs, rRNAs and tRNAs have already been well characterized. In the schematic diagram of a protein (bottom), the blue circles represent amino acids coded by unmodified codons and the red circles (with question marks) represent amino acids coded by pseudouridylated codons. Although it is known that the pseudouridylation of nonsense (stop) codons can result in the suppression of translation termination, it is not clear whether pseudouridylation of sense codons will lead to changes in coding specificity.

Figure 3. The occurrence and function of pseudouridines in various eukaryotic RNAs

Pseudouridines have been identified in a range of eukaryotic RNAs. Shown are typical secondary structures of the substrates (dark arrows) and potential substrates (light arrows) of the two types of pseudouridylation machineries — box H/ACA ribonucleoproteins (RNPs) and RNA-independent (stand-alone) pseudouridine synthases (PUSs). Also indicated are the known functions of the RNA substrates and of some pseudouridines in certain RNAs (unknown functions are denoted by question marks). rRNA, ribosomal RNA; RNase MRP RNA, ribonuclease mitochondrial RNA-processing RNA; snRNA, small nuclear RNA.