Two new sequencing methods may just make your day if you happen to be bidding for glorious insights into the epitranscriptome. Inspired by the bisulfite conversion used to assay DNA methylation, BID-seq and GLORI now enable single-nucleotide transcriptome-wide profiling of two crucial RNA modifications – N6-methyladenosine (m6A) and pseudouridine (the C5-glycoside isomer of uridine).
RNA modifications such as m6A and pseudouridine act to regulate protein synthesis; however, studies have also linked perturbed RNA modifications with pathological conditions. Understanding RNA modification dynamics may improve our fundamental understanding of this epitranscriptomic control mechanism and inform on disease development/treatment; however, we still require single-nucleotide resolution techniques to precisely measure RNA modifications across the transcriptome.
BID-seq adapts RNA bisulfite-sequencing to specifically modify target sites, resulting in site-specific deletions that allow the quantitative detection of pseudouridine levels. Meanwhile, GLORI supports the sequencing of m6A-modified sites by deaminating non-modified adenosines to inosine (with adenosines detected during reverse transcription identified as m6A) to enable m6A detection and quantification.
BID-seq: Betting on Pseudouridine!
Researchers led by Qing Dai, Li-Sheng Zhang, and Chuan He (University of Chicago) suggest we shouldn’t hedge our bets when choosing how to analyze transcriptome-wide pseudouridine dynamics. Instead, the authors wager that we should bet on their robust, quantitative pseudouridine mapping platform – BID-seq (bisulfite-induced deletion sequencing) – that builds on previous RNA bisulfite-sequencing methods.
Interestingly, like inosine, pseudouridine forms alternative base pairs and can ‘recode’ mRNAs. Studies have reported the translation of pseudouridine-modified mRNAs into multiple peptides via the induction of a point mutation or stop-codon read-through. Can BID-seq now tell us more about the impact of pseudouridine?
Dai, Zhang, Sun, Pajdzik, and colleagues report their new method, where pH adjustment of the bisulfite reaction improves pseudouridine-modified base deletion efficiency and inhibits side reactions to create a BID-seq protocol that quantitatively evaluates pseudouridine using only 10–20 ng input RNA:
- BID-seq detects alterations of pseudouridine modifications after the knockdown of individual pseudouridine synthetases enzymes in human cancer cells
- Hundreds of pseudouridine sites display occupation and conservation between mouse/human cells and mouse tissue
- Pseudouridine enrichment occurs in mRNA coding regions, suggesting that they influence protein synthesis
- Mouse tissues contain more pseudouridine-modified mRNA sites than human cell lines, with highly pseudouridine-modified transcripts displaying higher abundance and tissue-specific features
- Global analyses of mRNA half-lives reveal that pseudouridine modestly stabilizes transcripts
- Pseudouridine installed by TRUB1 (TruB Pseudouridine Synthase Family Member 1), the primary “writer,” prompts transcript stabilization in human cancer cells
- Investigations of mRNA recoding reveal that pseudouridine-modified stop codons exist in over 100 tissue transcripts
- Recoding stop codons into sense codons produce extended proteins from half of the modified transcripts
- Translational stop-codon read-through levels in individual modified mRNAs vary between transcripts and tissue (ranging from 3% to 35%)
Overall, the newly developed BID-seq provides a qualitative view of pseudouridine occupancy throughout the transcriptome but also demonstrates the existence of context-dependent pseudouridine-induced recoding of mRNA sequences. Don’t bet on any other technique to support findings like this!
GLORI-ous New Insight into m6A
Bidding for more glory from your study of RNA modifications? Well, put on a happy face, as researchers led by Chengqi Yi and Jing Wang (Peking University) describe GLORI (glyoxal and nitrite-mediated deamination of unmethylated adenosines). In GLORI, the deamination of unmethylated adenosines (to inosine) leaves m6A bases intact, allowing their detection by sequencing in a high-throughput single-base-resolution manner and permitting quantitative transcriptome-wide m6A mapping. Glyoxal catalyzes the conversion of adenosine to inosine and enables high-efficiency adenosine deamination under mild conditions that minimize unwanted side-conversion reactions. Superb!
Let’s hear all the details from this GLORI-ous new study from Liu and colleagues:
- GLORI detected more than 175,000 m6A sites in immortalized human embryonic kidney cells at single-base resolution in an unbiased, accurate, and highly-reproducible manner
- Analysis of m6A levels using GLORI suggests that most m6A-containing mRNAs possess multiple m6A-modified sites
- Around 33% of m6A occurs within modification “clusters” characterized by higher methylation levels with relative importance in gene expression regulation, reminiscent of 5mC DNA methylation in CpG islands
- High-level m6A modification decreases transcript half-life and reduces translation efficiency
- Heat shock and hypoxia dynamically induce alterations to m6A levels at between 4.8–11% of sites, which suggests that altering m6A levels may regulate gene expression in response to cell stress
Even though we already possess various tools that support m6A mapping, GLORI represents the first technique that allows the absolute quantification of transcriptome-wide m6A levels at single-base resolution in an unbiased and convenient manner, thereby supporting investigations into the role of this modification in biological regulation.
What Happens in the Epitranscriptome After the Celebrations Die Down?
The results of this BID-seq for GLORI may make you want to celebrate, given that these tools now provide the necessary quantification and resolution to move research into high throughput mechanistic studies of individual sites; however, the authors note limitations associated with these new approaches. These include a need for more precision at specific sequence contexts due to the elevated background and non-linear calibration curves and the need to develop robust algorithms to improve the calling of mRNA modification states.
For more on the future of mapping mRNA modifications, read more about BID-seq and pseudouridine at Nature Biotechnology, October 2022 and GLORI and m6A at Nature Biotechnology, October 2022.