Where you spend your time away from work – a lake-side cottage far from the hustle and bustle or a party pad in the bohemian part of town – is a crucial decision. Now, new research on roaming RNAs demonstrates that when you call the epitranscriptome your home, the age-old rule “location, location, location” also holds true for post-transcriptional modification and has biological significance by impacting translation.
A team of cunning cartographers led by Shalini Oberdoerffer (National Cancer Institute, NIH) previously reported an acetylated RNA immunoprecipitation method and demonstrated how the N4-acetylcytidine (ac4C) modification in mRNA coding sequences enhanced translational elongation by improving interactions with transfer RNAs. Interestingly, these mapping results also uncovered unexplained positional biases – ac4C accumulates near the 5′ ends (near translation start sites) and in the 5′ UTRs of transcripts, although no relationship to mRNA expression existed. In their follow-up study, this geographically gifted team now underscores a site-specific role for acetylation within mRNA 5′ UTRs in the regulation of translation initiation. Specifically, the team mapped the ac4C epitranscriptome at base resolution by coupling paired-end sequencing to sodium borohydride reduction of ac4C to tetrahydro-ac4C, which induces C to T mismatches during first-strand cDNA synthesis (RedaC:T-seq).
Let’s hear from Arango, Sturgill, and colleagues on the importance of the location, location, location of acetylation to mRNA translation:
- The team first validated RedaC:T-seq by comparing their results to acetylated RNA immunoprecipitation sequencing
- Analyses using systems for the controlled analysis of mRNA translation and HeLa cells demonstrate that ac4C modification within RNA 5′ UTRs decreases translation initiation at annotated start codons, thereby impacting protein synthesis
- Meanwhile, RNA 5′ UTR ac4C prompts a concomitant increase in translation at alternative initiation sites that encompass non-canonical sequences, providing evidence of competitive inhibition of annotated start codons
- Overall, ac4C within 5′ UTRs inhibits canonical translation initiation sites and promotes non-optimal initiation
- Mechanistically, ac4C influences translational initiation by modulating base-pair interactions
- ac4C within 5′ UTRs induces the generation of structures that repress translation initiation by inhibiting preinitiation complex scanning and directly inhibits translation by interfering with interactions of modified mRNAs with the initiator methionine transfer RNA
Overall, this exciting new study describes the epitransciptomic regulation of mRNA translation initiation and, more specifically, underscores the importance of the site-specific N4-acetylcytidine modification. Future studies will focus on additional cells and tissues and optimizing methodologies for quantitatively assessing ac4C at base resolution.
For more on how the location, location, location of epitranscriptomic modifications can influence translation, see Molecular Cell, June 2022.