Small RNAs are important transcriptional and post-transcriptional regulators of gene expression. Many classes of small RNAs have been discovered, each carrying out specialized functions. siRNAs and miRNAs are best studies. siRNAs function in the process of RNAi and are thought to defend the genome against molecular parasites. They have become widely used tools in biomedical research. miRNAs are abundant and conserved; they form a class of endogenous small RNAs that fine-tunes gene expression. Most, if not all, small RNAs function through association with members of the Argonaute protein family. This thesis focuses on the function of small RNAs and Argonaute proteins in RNAi and the miRNA pathway. To understand the role of miRNAs in development, we aimed to catalogue the zebrafish miRNAs and analyze their expression. By sequencing miRNA libraries from 5-day-old zebrafish larvae and adult zebrafish brain, we found 139 known and 66 new miRNAs. We analyzed the temporal and spatial expression patterns of 67 miRNAs by whole mount in situ hybridization and northern blot analysis. Most miRNAs are expressed during later stages of development, often in a tissue specific manner. Most newly discovered miRNAs have low expression levels and are less conserved in other vertebrate species. We also analyzed the connection between the miRNAs and Argonaute proteins in C. elegans. The Argonaute proteins required for the miRNA pathway are ALG-1 and ALG-2. We used massively parallel sequencing to analyze the spectrum of small RNAs associated with ALG-1. We found that this protein almost exclusively binds to miRNAs. In addition to 84 known miRNAs, we found ten novel miRNAs. RNAi is an amplified process in C. elegans. In the amplification step, RNA-directed RNA polymerases (RdRPs) produce secondary siRNAs, which we cloned from transgenic lines expressing a single primary siRNA. We found that RdRPs perform unprimed RNA synthesis to generate secondary siRNAs, and that mRNAs which are not cleaved by the primary siRNA-Argonaute complex can be used as substrates. Secondary siRNAs are only of antisense polarity, carry 5' di- or triphosphates, and are only in the minority associated with RDE-1, the primary siRNA-binding Argonaute protein. Therefore, secondary siRNAs represent a distinct class of small RNAs. siRNAs and miRNAs are both processed from dsRNA-precursors by the dsRNase DCR-1. We investigated how RNAi and the miRNA pathway are mechanistically separated. We showed that precursors of small RNAs contain structural features that direct the small RNAs into the RNAi or the miRNA pathway. Small RNAs expressed from hairpin precursors with a fully matching stem are recognized as siRNAs and bound by RDE-1. A one- to three-nucleotide mismatch at various positions in the stem of the precursor directs the small RNAs into the miRNA pathway, as these small RNAs are in majority bound to ALG-1. The Argonaute proteins to which the small RNAs are bound determine the silencing mode, and no functional overlap between RDE-1 and ALG-1 was detected. We also provide evidence that RNAi is slicer-independent in C. elegans and analyze RDE-1-associated small RNAs as an approach to the endogenous role of RNAi
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