Argonautes are the main components of effector complexes in RNAi and are responsible for the small RNA-guided destruction of mRNAs during this process. In addition to this key role in RNAi, Argonautes also function in related pathways, including translational and transcriptional gene silencing, heterochromatin formation and centromeric function, and chromosome elimination in Tetrahymena. C. elegans has 27 Argonaute family members, and our lab has focused on understanding the roles of these individual proteins in various silencing pathways. One Argonaute F20D12.1, now
csr-1 (for chromosome segregation and RNAi deficient), is particularly intriguing, as it plays roles in fertility, chromosome segregation, and RNAi. We have rescued the sterility and embryonic lethality of
csr-1 mutants with transgenes encoding GFP- and 3xFlag-tagged CSR-1. In these rescued strains, we observe a partial rescue of the germline RNAi deficiency of
csr-1 mutants. However, expression of CSR-1 is insufficient to rescue other germline rde Argonaute mutants (such as
ppw-1), the
rde-1 mutant that acts in the initiation of RNAi, or a multiple Argonaute mutant (MAGO) that is deficient in the downstream steps of RNAi. These results suggest that CSR-1 plays a distinct role in germline RNAi, and experiments are ongoing to determine for which steps in germline RNAi CSR-1 is required. In addition to the RNAi defect, mutation of
csr-1 leads to defects in germline progression and chromosome segregation during early embryonic divisions. These phenotypes may indicate a defect in silencing pathways necessary for proper chromatin formation, and may be similar to a silencing process in S. pombe that requires a sole yeast Argonaute and is necessary for the proper formation of centromeric heterochromatin. In order to find small RNAs involved with CSR-1 in this process, we have taken a candidate approach using Northern blotting, as well as an unbiased search using 454 Sequencing of small RNAs. These studies have revealed a class of small RNAs that is missing in the
csr-1 mutant. Our current analyses are focused on understanding the roles of these small RNAs in germline development, and identifying additional small RNAs with which CSR-1 interacts. In addition to the small RNAs involved in CSR-1-mediated silencing, we are using IP/mass spec approaches to identify CSR-1 interacting proteins and gain an understanding of the functions of CSR-1, including a potential role in centromere formation.