From Greta Thunberg leading global climate protests to Katie Bouman capturing the first picture of a black hole; sometimes an emerging star can create enormous waves on the world scene. In the realm of epigenetics, the “humble” N 7-methylguanosine (m7G) modification forms the eukaryotic mRNA cap, a structure that regulates mRNA export, translation, and splicing, and also appears within transfer and ribosomal RNAs. Excitingly, two studies now establish m7G as an emerging star of the epitranscriptomic scene by describing the presence of this modification within human mRNAs and micro (mi)RNAs.
The highly stable nature of m7G had precluded the detailed assessment of this modification in the past; however, both featured articles report a novel method (Borohydride Reduction Sequencing or BoRed-seq) that selectively converts internal m7G sites on the decapped RNA into abasic sites (via sodium borohydride exposure) that are then covalently bound by a biotin-coupled aldehyde reactive probe. After pull-down with streptavidin beads and reverse transcription, the biotinylated sites are identified as internal m7G sites via high-throughput sequencing.
Its What’s on the Inside that Counts for m7G-modified mRNA
No one has ever accused epigenetics of being shallow and vapid, and the first of our featured articles from the lab of Chuan He (The University of Chicago, USA) establishes that for the emerging epitranscriptomic star m7G, it really is what is on the inside (of mRNA) that counts. The modest team of researchers placed all the trappings of the high life to one side and employed a range of robust techniques to discover more about the inner mRNA beauty of m7G:
- Mass spectrometry-based quantification revealed the presence of internal m7G sites in decapped mRNA derived from a range of normal and cancer cell lines
- Confirmation of this finding by m7G-methylated RNA immunoprecipitation sequencing (MeRIP-seq) also demonstrated similar distribution patterns and high conservation between human and mouse
- BoRed-seq generated the first base-resolution maps of m7G in human mRNA
- Internal m7G with high modification levels display GA-enriched motifs and occur near the start and stop codon, which suggests a functional role
- siRNA knockdown and PAR-CLIP assays (employed to detect the interaction between specific RNAs and proteins) revealed METTL1/WDR4 as the critical methyltransferase complex for a subset of m7G modifications within mRNAs
- m7G may facilitate translation of modified mRNAs, as knockdown of METTL1 decreases the translation efficiency of transcripts that contain METTL1-affected hypomethylated sites
Overall, the authors hypothesize that m7G may influence mRNA structure (see the next study for more on this!) or mediate protein-RNA interactions and anticipate that their work will provide a resource for future research into the links between m7G levels and biological processes and human diseases. Let’s hope this new found fame will not influence the future trajectory of m7G, an exciting new star emerging onto the epitranscriptomic scene.
Internal m7G Modification of miRNAs Stops Cells in their Tracks
In the second of our m7G-based articles, researchers from the lab of Tony Kouzarides at the University of Cambridge (UK) also employ the unique chemical reactivity of m7G and related techniques to generate RNA maps; however, the talented team chose to map the presence of this emerging epitranscriptomic star on miRNA, and what they discovered stopped them in their tracks:
- m7G MeRIP-seq and BoRed-seq with decapped cancer cell RNA revealed the presence of a subset of mature miRNAs with internal m7G
- The level of m7G modification on a miRNA displays no correlation with its expression level, but m7G does represent a general and conserved modification
- This subset includes tumor-suppressive miRNAs, including let-7, which is known to inhibit cell migration
- The METTL1 methyltransferase mediates m7G methylation of miRNA, where the loss of this catalytic activity correlates with altered gene expression and the increased migration of cancer cells
- Mass spectrometry-based analysis of purified
RNA revealed a single m7G modification within the let-7e-5p primary (pri-) miRNA
- This modification facilitates let-7 miRNA processing by disrupting the formation of an inhibitory secondary structure (G-quadruplex), thereby impelling the formation of a canonical stem-loop
- This conformational change promotes the processing of the pri-miRNA into precursor (pre-) miRNA, permitting miRNA maturation (cleavage by DICER into the final miRNA duplex) and the suppression of lung cancer cell migration
Overall, the authors suggest that m7G may act to disrupt RNA structures that inhibit their translation and stop cells dead in their tracks, leaving them to anticipate that their findings may open new therapeutic avenues in miRNA-related disease.
In summary, both studies make an excellent case for the emergence of m7G from a humble past into a bright epitranscriptomic future! For more on the inner beauty of m7G see the article by Zhang and colleagues (Molecular Cell, April 2019) and migrate quickly over to the article from Pandolfini and colleagues (Molecular Cell, April 2019) for all the show-stopping details in m7G.