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Molecular Characterization of the microRNA Life-Cycle

Final 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 largest 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.
This project focused on the least understood aspect in small RNA-mediated gene silencing, the regulation of microRNA homeostasis. Our goal was 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. To this end, we generated a comprehensive atlas of post-transcriptional modifications in small RNAs, revealing that microRNAs and their precursors are frequently modified at their 3´end by uridylation and adenylation; and we determined the origin and biological function of these modifications, which play a crucial role in the regulation of microRNA maturation. Further studies revealed that RNA uridylation also plays a crucial role in the regulation of coding and non-coding RNAs and therefore establishes a novel layer in the regulation of gene expression.
We also developed a novel method, called SLAMseq, which enables to follow the fate of RNA molecules inside living cells. By combining SLAMseq with massive parallel sequencing of miRNAs, we determined the intracellular kinetics of miRNA biogenesis, their loading into ribonucleoprotein complexes and their decay. Our studies revealed that miRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells. Together, our studies provided the first comprehensive view on the intracellular kinetics of small RNA silencing pathways. Beyond the proposed work, we have also applied SLAMseq to dissect gene regulatory pathways in cancer, unraveling the function of the principal oncogenic transcription factor Myc. The project resulted in a patent application. The underlying SLAMseq technology was licensed and commercialized world-wide as “SLAMseq metabolic RNA sequencing kit” by Lexogen GmbH.