Mechanism and biology of RNA silencing in flies and mammals
Small silencing RNAs regulate gene expression in nearly all eukaryotes and have enormous biotechnological and therapeutic potential. MicroRNAs (miRNAs) belong to the largest family of trans-acting gene regulatory molecules in multicellular organisms. In flies and mammals, they control more than a half of the protein-coding transcriptome, and act as key regulators of organismal development, physiology, and disease. We are interested in understanding the molecular mechanisms that govern small RNA-mediated gene silencing in flies and mammals.
We aim to investigate the molecular processes that regulate the production of small RNAs, their assembly into ribonucleoprotein complexes, and their sequence-specific decay. Our goal is to define the principles that establish and maintain small RNA profiles in a given tissue or cell type in order to understand the molecular mechanisms that regulate miRNA homeostasis. To do so we utilize a combination of Drosophila genetics and biochemistry, and RNomics. The hypotheses derived from our studies in flies are directly tested in regard of their applicability in mammals.
Regulation of microRNA biogenesis
The post-transcriptional addition of uridine nucleotides to the 3´ end 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. Recently, we identified Tailor, an uridylyltransferase that is required for the majority of 3´ end modifications of miRNAs in Drosophila which predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3´ overhang of miRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, a class of non-canonical miRNA precursors generated directly by splicing of introns from messenger RNAs. Mirtron selectivity is explained by the primary sequence specificity of Tailor, selecting substrates ending with a 3´ guanosine, a hallmark of introns. In contrast to mirtrons, conserved Drosophila precursor miRNAs are significantly depleted in 3´ guanosine, thus evading regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor miRNA 3´ ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of miRNAs in Drosophila.
Target RNA-directed small RNA decay
Small RNAs guide Argonaute proteins to complementary sequences within mRNAs. In animals, miRNAs typically show just partial complementarity to the targets they regulate. We recently found that high complementarity between miRNAs and their targets causes small RNAs to decay. This occurs in a process that involves the addition of non-templated nucleotides to the 3´ end of small RNAs (tailing) and their 3´ to 5´ exonucleolytic degradation (trimming) (Figure 2). Notably, some herpes viruses highjack the cellular miRNA decay machinery to trigger the destruction of host miRNAs in order to counteract the antiviral function of the microRNA pathway. We aim to characterize the molecular details of this novel miRNA decay pathway, identify its enzymatic components, and determine the biological function of the pathway. Our hypothesis is that mRNAs not only serve as targets for miRNA-mediated gene regulation, but also influence the abundance, and therefore the function of miRNAs themselves.
Therapeutic miRNA inhibition
The sequence-specific decay of miRNAs harbors considerable therapeutic potential. For instance, the inhibition of miR-122 – a liver-specific regulator of lipid metabolism – reduces serum cholesterol levels and interferes with replication of the hepatitis C virus (HCV). We recently developed a novel approach for efficient long-term inhibition of miRNA function in vivo in mice.
The expression of tough decoy RNAs (TuDs, Fig. 3) – structured RNA polymerase III transcripts with accessible and?highly complementary miRNA target sites – efficiently triggers miRNA decay by inducing the tailing and trimming pathway in? cultured human cells and in vivo in mice, after recombinant adeno-associated virus (rAAV) vector delivery. rAAV-mediated miRNA inhibition provides a simple means of studying miRNA function in adult mammals and may serve as a treatment for dyslipidemia and other miRNA-related human diseases.