Epitranscriptomic regulation of non-coding RNAs

Stefan Ludwig Ameres1,
1IMBA, Austria 

For RNA to fulfil its essential function within the cellular environment, numerous chemical modifications have evolved to sculpt its physical and functional interactions. Although more than hundred types of RNA modifications have built the descriptive foundation of what is referred to as the epitranscriptome, their mode of action remains largely unknown. I will present our results on the function of chemical RNA modifications at the intersection of small RNA silencing pathways and general RNA metabolism.
Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. We identified the first RNA- specific uridylytransferase that is required for the majority of 3′ end modifications of microRNAs in Drosophila and predominantly tar- gets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3′ overhang of microRNA hairpins, which regu- lates processing by Dicer and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3′ guanosine. In contrast to mirtrons, con- served Drosophila precursor microRNAs are significantly depleted in 3′ guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3′ ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila. Our data also provide an atlas of post-transcriptional modifications in small RNAs and their precursors in flies, providing a framework for understanding the epitranscriptomic regulation of small RNA biogenesis and function.
We could also show that uridylation in flies triggers the pro- cessive 3′-to-5′ exoribonucleolytic decay via the ribonuclease II/R enzyme CG16940, a homolog of the human Perlman syn- drome exoribonuclease Dis3l2. Together with the TUTase Tailor, dmDis3l2 forms a stable cytoplasmic uridylation-triggered RNA processing (TRUMP) complex, that functionally cooperates in the degradation of structured RNAs in vitro, providing a molecular explanation for the inhibition of mirtron maturation in flies. RNA- immunoprecipitation and high-throughput sequencing reveals a variety of TRUMP complex substrates, including long non-coding RNA, such as rRNA, the essential RNase MRP and the signal recogni- tion particle RNA 7SL. Together with high-throughput biochemical characterization of dmDis3l2 and bacterial RNase R our results imply a conserved molecular function of RNase II/R enzymes as ‘readers’ of destabilizing post-transcriptional marks–uridylation in eukaryotes and adenylation in prokaryotes–that play important roles in non-coding RNA surveillance.

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