PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia

Abstract

Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase.Supplemental material is available at http://www.genesdev.org.

Subtraction by addition: domesticated transposases in programmed DNA elimination: Figure 1.

“…This new report from Baudry et al (2009) shows that Paramecium uses what was once a transposase from a piggyBac element to reorganize its somatic genome. When this putative endogenous transposase was fused to green fluorescent protein, it localized to developing MACs where DNA elimination occurs, leading Baudry et al (2009) to call this gene PiggyMac (PGM). To further assess the possibility that Pgm participated in DNA elimination, the group knocked down PGM levels by feeding Paramecium bacterial cells expressing PGM-specific dsRNA prior to inducing autogamy.…”

“…To further assess the possibility that Pgm participated in DNA elimination, the group knocked down PGM levels by feeding Paramecium bacterial cells expressing PGM-specific dsRNA prior to inducing autogamy. This treatment resulted in a 20-fold reduction in progeny production and thus revealed that the gene product has a critical function during development (Baudry et al 2009). …”

“…To test this possibility, Baudry et al (2009) used ligation-mediated PCR to assay the ends of a known IES for the appearance of double-strand breaks (DSBs) that normally occur during DNA elimination. In the PGM knockdown cells, they failed to detect breaks that were readily observed in wildtype cells.…”

“…To examine the timing of Pgm function, Baudry et al (2009) exploited the fact that mating cell pairs separate and begin refeeding before the developing MACs carry out DNA elimination (Berger 1973). Feeding cells PGM dsRNA early (before conjugation starts) or late (after pairs separated) interfered with normal development.…”

“…The results of several recent studies suggest that the recognition of DNA to be packaged into heterochromatin may be a precursor to genome-wide DNA elimination. A report from the research group of Betermier (Baudry et al 2009) in this issue of Genes & Development reveals that a domesticated piggyBac transposase has been recruited to carry out programmed DNA elimination in the ciliate Paramecium tetraurelia. Previous work in Paramecium and related ciliates has shown that an RNAi-related machinery is also essential for these DNA rearrangements, providing a key link between DNA elimination and heterochromatin formation (for review, see Yao and Chao 2005;Duharcourt et al 2009).…”