Pseudouridines in RNAs: switching atoms means shifting paradigms.

The structure, stability, and function of various coding and noncoding RNAs are influenced by chemical modifications. Pseudouridine (Ψ) is one of the most abundant post-transcriptional RNA base modifications and has been detected at individual positions in tRNAs, rRNAs, mRNAs, and snRNAs, which are referred to as Ψ-sites. By allowing formation of additional bonds with neighboring atoms, Ψ strengthens RNA-RNA and RNA-protein interactions. Although many aspects of the underlying modification reactions remain unclear, the advent of new transcriptome-wide methods to quantitatively detect Ψ-sites has recently changed our perception of the functional roles and importance of Ψ. For instance, it is now clear that the occurrence of Ψs appears to be directly linked to the lifetime and the translation efficiency of a given mRNA molecule. Furthermore, the administration of Ψ-containing RNAs reduces innate immune responses, which appears strikingly advantageous for the development of generations of mRNA-based vaccines. In this review, we aim to comprehensively summarize recent discoveries that highlight the impact of Ψ on various types of RNAs and outline possible novel biomedical applications of Ψ.

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.

Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding RNAs (ncRNAs), including rRNAs, tRNAs, and snRNAs, where it contributes ∼7% of the total uridine level. However, Ψ constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of Ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based Ψ mapping technique called photo-crosslinking-assisted Ψ sequencing (PA-Ψ-seq) and use it to map Ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe 293T-derived cell lines in which human PUS enzymes previously reported to add Ψ residues to human mRNAs, specifically PUS1, PUS7, and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of Ψ addition on cellular mRNAs to each of these three PUS enzymes, Ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7, or TRUB1 function did not significantly reduce the level of Ψ residues detected on total human mRNA below the ∼0.1% level seen in wild-type cells, thus implying that the PUS enzyme(s) that adds the bulk of Ψ residues to human mRNAs remains to be defined.

Application of a recombinant Fab fragment from a phage display library for sensitive detection of a target antigen by an inhibition ELISA system.

We have found that the recombinant Fab (rFab) produced by phage display system was detectable for a target antigen more sensitive than the parental monoclonal antibody (MoAb). The Fab phage display library was constructed from hybridoma cells producing APU-6 MoAb specific for a modified nucleoside, pseudouridine that have been studied as a urinary marker for malignancy. Fab-displayed phage clones were screened by a direct ELISA, and the single positive clone was finally obtained. Although the reaction pattern of rFab against pseudouridine and uridine was almost identical to that of MoAb, detection sensitivity of rFab was approximately 30 times higher than that of MoAb. Since the sensitivity of rFab was almost identical to that of Fab fragment prepared by papain digestion of MoAb, the increased sensitivity is considered to be the nature of Fab fragment. The sensitivity of established assay system was sufficient for quantitative determination of serum pseudouridine levels in healthy individuals and cancer patients. This procedure may be applicable for improvement of detection sensitivity of a MoAb-based inhibition ELISA system for drugs or low molecular weight compounds.

An immunohistochemical analysis for cancer of the esophagus using monoclonal antibodies specific for modified nucleosides.

BACKGROUND: Modified nucleosides such as 1-methyl-adenosine and pseudouridine exist as minute components of transfer ribonucleic acid (tRNA) and are excreted in the urine in large amounts in the presence of malignancy. Although use of these modified nucleosides as tumor markers has long been studied and many reports have detailed their relationship with malignant tumors and the urinary excretion of various modified nucleosides, there have been no reports on modified nucleosides in esophageal carcinoma. METHODS: Monoclonal antibody patterns against 1-methyladenosine and pseudouridine were studied in esophageal carcinoma, freshly resected esophageal carcinoma tissue specimens fixed in 10% neutral formaldehyde solution, embedded in paraffin, and sectioned for immunohistochemical study. Inhibition enzyme-linked immunosorbent assay (ELISA) was used to examine urinary excretion of these modified nucleosides in patients with esophageal carcinoma. RESULTS: Although rare in normal esophageal epithelium, these modified nucleosides were strongly stained in esophageal carcinoma cells. Most carcinoma cells exhibited a cytoplasmic pattern, although some cells at the infiltrating edge displayed a nuclear pattern. These modified nucleosides were intensely imaged in 11 of 12 cultured esophageal cell lines, the exception being one line that had a much longer doubling time. Using ELISA, urinary excretion of these modified nucleosides was found to be significantly higher in patients with esophageal carcinoma than in healthy subjects; such excretion correlated with carcinoma size and stage and tended to decrease after treatment. CONCLUSIONS: These findings indicate that the modified nucleosides 1-methyladenosine and pseudouridine may be useful as tumor markers for esophageal carcinoma.

Monoclonal antibody to pseudouridine used to develop a radioimmunoassay.

Pseudouridine, a component of tRNA, was modified to yield the derivatives: succinyl, palmitoyl pseudouridine, and protein conjugates. These derivatives were used in preparation of monoclonal antibodies specifically directed to pseudouridine. MAbs from three hybridomas (OAL 881, 812 and 814) were established and shown to be directed to pseudouridine, uridine and uracil. OAL 881 was equally reactive with the three substrates, OAL 812 showed the same reactivity for pseudouridine and uracil, while OAL 814 showed a reactivity mainly for pseudouridine. MAb OAL 814 was used in a radioimmunoassay (RIA) system unique for pseudouridine. A good dose-response curve was observed in the range between 31.3 and 2000 nmol/ml. Intra- and inter-assay CV values were below 4.1% and 7.4% respectively, and good results were obtained from recovery of added material and dilution tests. The ratio of pseudouridine/creatinin in urine was significantly higher in patients with cancer than in normal subjects.

Detection of elevated amounts of urinary pseudouridine in cancer patients by use of a monoclonal antibody.

Preparation of anti-pseudouridine monoclonal antibodies (MoAbs) and their applications for the quantitation of urinary pseudouridine in cancer patients are described. Seven MoAbs were selected. Five MoAbs were specific for pseudouridine and two MoAbs were cross-reactive with uridine. The most specific antibody, APU-6, was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary pseudouridine. Sensitivity was in the picomole range and the accuracy was nearly equal to that of the high performance liquid chromatography (HPLC) assay. The amount of pseudouridine in the urine of 28 healthy donors was 31.17 +/- 9.94 nmol/mumol creatinine. In 55% (35/63) of patients with cancer, urinary pseudouridine was elevated above the normal mean + 2 SD (51.04 nmol/mumol creatinine). Particularly, all of the patients (15/15) with leukemia and lymphoma had elevated levels of pseudouridine. These results suggest that urinary pseudouridine might be useful as a marker for leukemia and lymphoma.