In the Drosophila germ line, PIWI-interacting RNAs (piRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences.
We examined the genetic requirements for the biogenesis and function of piRNAs in both female and male germ line. We found that piRNAs function through the PIWI, rather than the AGO, family Argonaute proteins, and the production of piRNAs requires neither microRNA (miRNA) nor small interfering RNA (siRNA) pathway machinery. These findings allowed the discovery of the third conserved small RNA silencing pathway, which is distinct from both the miRNA and RNAi pathways in its mechanisms of biogenesis and function.
We also found piRNAs in flies are modified. We determined that the chemical structure of the 3´-terminal modification is a 2´-O-methyl group, and also demonstrated that the same modification occurs on the 3´ termini of siRNAs in flies. Furthermore, we identified the RNA methyltransferase Drosophila Hen1, which catalyzes 2´-O-methylation on both siRNAs and piRNAs. Our data suggest that 2´-O-methylation by Hen1 is the final step of biogenesis of both the siRNA pathway and piRNA pathway.
Studies from the Hannon Lab and the Siomi Lab suggest a ping-pong amplification loop for piRNA biogenesis and function in the Drosophila germline. In this model, an antisense piRNA, bound to Aubergine or Piwi, triggers production of a sense piRNA bound to the PIWI protein Argonaute3 (Ago3). In turn, the new piRNA is envisioned to produce a second antisense piRNA. We isolated the loss-of-function mutations in ago3, allowing a direct genetic test of this model. We found that Ago3 acts to amplify piRNA pools and to enforce on them an antisense bias, increasing the number of piRNAs that can act to silence transposons. Moreover, we also discovered a second Ago3-independent piRNA pathway in somatic ovarian follicle cells, suggesting a role for piRNAs beyond the germ line.
| Identifer | oai:union.ndltd.org:umassmed.edu/oai:escholarship.umassmed.edu:gsbs_diss-1530 |
| Date | 05 April 2011 |
| Creators | Li, Chengjian |
| Publisher | eScholarship@UMassChan |
| Source Sets | University of Massachusetts Medical School |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Morningside Graduate School of Biomedical Sciences Dissertations and Theses |
| Rights | Copyright is held by the author, with all rights reserved., select |
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