Regulation of genome expression is essential for cell proliferation, differentiation, development and viability. Small RNA silencing pathways are involved in the cellular control of gene expression and in protection of the genome against mobile repetitive DNA sequences, retroelements and transposons. Small RNAs interact with target RNAs and promote chromatin modifications, translational inhibition or degradation of complementary RNAs. We have uncovered new class of small RNAs in fission yeast, priRNAs, which are generated independently of Dicer. priRNAs are likely to be involved in triggering of siRNA amplification and heterochromatin assembly within centromeric repeats. In first aim, using small RNA sequencing and transcriptomic approaches, combined with biochemistry and functional assays, we will determine biogenesis and function of noncentromeric priRNAs. Specifically, Argonaute-dependent 3' end formation of priRNAs will be studied, a process conserved in piRNA biogenesis in higher eukaryotes. In second aim, we will study what recruits RNAi to transposable and repetitive elements. Furthermore, we will study RNA susceptibility to RNA interference. In third aim, we will use cryo electron microscopy to determine structure of chromodomain protein Chp1 bound to nucleosomes with histone 3 lysine 9 methylated. We will extend this study on other RNAi and heterochromatin complexes. Defects in small RNA-mediated regulation of genome expression have been described in several human cancers. Fundamental understanding of the role of small RNAs in gene regulation will help us understand why some cells loose their identity and turn into cancer cells.