Why U matters: detection and functions of pseudouridine modifications in mRNAs

Abstract

The uridine modifications pseudouridine (Ψ), dihydrouridine, and 5-methyluridine are present in eukaryotic mRNAs. Many uridine-modifying enzymes are associated with human disease, underscoring the importance of uncovering the functions of uridine modifications in mRNAs. These modified uridines have chemical properties distinct from those of canonical uridines, which impact RNA structure and RNA-protein interactions. Ψ, the most abundant of these uridine modifications, is present across (pre-)mRNAs. Recent work has shown that many Ψs are present at intermediate to high stoichiometries that are likely conducive to function and at locations that are poised to influence pre-/mRNA processing. Technological innovations and mechanistic investigations are unveiling the functions of uridine modifications in pre-mRNA splicing, translation, and mRNA stability, which are discussed in this review.

Keywords: mRNA modifications; mRNA stability; pre-mRNA processing; pseudouridine; splicing; translation.

Figure 1.. Uridine modifications in mRNA.

a) Distribution of uridine modification across mRNAs. b) Digestion of mRNAs followed by bulk nucleoside LC/MS is applied to determine the presence and absolute abundance of uridine modifications within mRNAs based on spectra intensity and mass shift of modified compared to unmodified nucleoside. c) Sequencing-based mapping tools reliant on chemical derivatization of modifications (CMC and bisulfite) and reverse transcriptase signatures (RT stops and deletions respectively) are used to identify the location, and with the aid of synthetic standards, the stoichiometry of uridine modifications transcriptome-wide. d) Direct RNA long-read sequencing with Nanopore Technology allows identification of co-occurring uridine modifications within full-length mRNAs based on signal differences (e.g. current intensity,) as modified compared to unmodified mRNAs translocate through Nanopores. e) Site-directed modifications to interrogate functions. Recently, engineered snoRNAs with base complementary to target sites of interest have allowed site-specific installation of individual pseudouridines in mRNA by the pseudouridine synthase DKC1. PUSs have been fused to dead RNA targeting CAS13 (dCAS13) to install Ψ at specific locations directed by guide RNAs (gRNA). Although some challenges remain, these and new technologies could represent tools to study the direct functions of individual modifications in mRNAs.

Figure 2.. Functions of pseudouridines in mRNAs.

Emerging evidence suggests that Ψ may function in mRNA processing and mRNA metabolism. Ψ in pre-mRNAs are added by pre-mRNA pseudouridine synthases such as PUS1 and PUS7. Pseudouridines are enriched within splice sites including the 3’ splice site (3’ ss) and the polypyrimidine tract (PPT), and can impact alternative splicing outcomes. Ψ in coding sequences, at the 1^st or 3^rd position in Phe codons can impact translation fidelity leading to recoding to amino acids Leu and Val. Ψ in UAG and UGA stop codons leads to stop codon readthrough and misincorporation of Gln and Arg. PUS1 has been shown to pseudouridylate a stop codon. TRUB1-dependent Ψs (in the context of the TRUB1 motif GUΨC) in mRNAs have been shown to stabilize mRNAs and loss of Ψ leads to destabilization. Future work investigating direct effects of individual modifications in mRNAs in cells and providing mechanistic insight are needed to clarify direct versus indirect functions of uridine modifications in mRNA and to reveal the extent to which mRNA modifications are used for gene regulation.

Figure I.

Structures of modified uridines.