The Madhani Lab is in the Department of Biochemistry and Biophysics at UCSF
ucsfmadhani lab

RNA-guided genome defense

(1) Recognition and silencing of transposable elements: a new function for the spliceosome

Transposable elements are an existential threat to life. How they are recognized and silenced is poorly understood. Using the yeast Cryptococcus neoformans, we have discovered a new RNA-based mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We found that intron-containing mRNA precursors template siRNA synthesis. We identified a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrated that it physically associates with the spliceosome. Furthermore, we observed that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrated that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme was also found to be necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense. We are investigating this exciting new pathway by which stalled spliceosomes produce silencing siRNAs. We are also examining how silencing of transposons occurs in this system using the powerful whole-genome tools available in this haploid budding yeast.

genome defense

(2) Recognition and silencing of repeats: a new RNA-guided pathway in fission yeast

Like transposons, simple repeat sequences can have a deleterious impact on organisms by causing mutations. Using the yeast Schizosaccharomyes pombe, we identified a new RNA-guided mechanism of silencing of pericentromeric repeats. In S. pombe, heterochromatin assembly on transcribed non-coding pericentromeric repeats requires both RNAi and RNAi-independent mechanisms. In Saccharomyces cerevisiae, which lacks repressive chromatin mark (H3K9Me), unstable ncRNAs are recognized by the RNA-binding protein Nrd1. We found that the S. pombe ortholog, Seb1, is associated with pericentromeric lncRNAs. Furthermore, we demonstrated that mutation of dcr1+ (Dicer) or seb1+ results in equivalent partial reductions of pericentromeric H3K9Me levels. Strikingly however, a double mutation eliminates this mark. We found that Seb1 functions independently of RNAi by recruiting NuRD-related chromatin-modifying complex SHREC. The discovery of an RNA-binding protein that mediates the action of a long ncRNA in chromatin silencing offers a unique opportunity to dissect the mechanistic underpinnings of RNA-controlled silencing. We anticipate that it will also lead to an understanding how the RNAi-dependent arm of chromatin silencing is regulated or "licensed" in fission yeast. Our aim is to identify the signals necessary and sufficient for RNA-guided, chromatin-based repeat silencing in S. pombe and to develop a detailed mechanistic understanding of their action. We anticipate that such knowledge will impact our understanding of long ncRNAs in mammalian systems.

RNA guided genome defense

genome defense research

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