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ERC

miRLIFE Report Summary

Project ID: 338252
Funded under: FP7-IDEAS-ERC
Country: Austria

Mid-Term Report Summary - MIRLIFE (Molecular Characterization of the microRNA Life-Cycle)

Small silencing RNAs regulate gene expression in nearly all eukaryotes and have enormous biotechnological and therapeutic potential. MicroRNAs belong to the larges family of trans-acting gene regulatory molecules in multicellular organisms. In flies and mammals, they control more than half of the protein-coding transcriptome, and act as key regulators of organismal development, physiology, and disease.

The ERC Starting Grant miRLIFE supports our efforts to study the molecular mechanisms that regulate microRNA homeostasis. We aim to understand how distinct small RNA profiles are established and maintained to coordinate the expression of more than half of all protein coding genes in flies and mammals. Our studies provide insight into the processes that regulate the function of miRNAs, determine possible causes for aberrant miRNA levels, that have been associated with human diseases, and provide guidelines how to efficiently inhibit miRNA function for analytical and therapeutic purposes. Understanding the molecular principles underlying the regulation of small RNA silencing will certainly also contribute to a better understanding of intracellular RNA metabolism and associated diseases.

Because of its genetic and biochemical tools, we use Drosophila melanogaster as a model organism. We employ a combination of bioinformatics, cell-free biochemical experiments, cell culture methods, and in vivo genetics. What we learn in flies we is test in vitro in mammalian cell extracts, in cultured human cell lines and in vivo in mice to identify where these processes are conserved and where they diverge.

Significant progress is summarized through the following central findings:

- RNA uridylation confines the emergence of microRNAs in Drosophila
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 targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3 overhang of microRNA hairpins, which regulates pro- cessing 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, conserved 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.

- A novel cytoplasmic RNA decay complex unravels the function of uridylation in non-coding RNA surveillance
The post-transcriptional addition of nucleotides to the 3 end of RNA regulates the maturation, function and stability of numerous RNA species in all domains of life. We could show that uridylation in flies triggers the processive 3-to-5 exoribonucleolytic decay via the ribonuclease II/R enzyme CG16940, a homolog of the human Perlman syndrome exo-ribonuclease 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 recognition 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 RNA surveillance.

Contact

Tanja Winkler, (Grants Manager)
Tel.: +43 1 79044 4410
Fax: +43 1 79871 53
E-mail
Record Number: 191569 / Last updated on: 2016-11-21