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.

Figure 1.

Developmentally regulated expression and cellular localization of the PiggyMac domesticated transposase. (A) Structure of the predicted Pgm protein and comparison with the piggyBac transposase from Trichoplusia ni. The catalytic D residues are indicated by asterisks. (Black boxes) Conserved core. The conserved Cys-rich domain is shown in orange, and the C-terminal coiled-coil domain (predicted by the Lupas algorithm) is shown in gray. (B, top two panels) Northern blot analysis of PGM and PtSPO11 mRNA levels during autogamy of 51new. (Bottom two panels) Ligation-mediated PCR (LMPCR) detection of DSBs at the left boundary of IESs 51A1835 and 51G4404 in the same autogamy time course (see Supplemental Fig. S2). (V) Vegetative cells. (C) Localization of the GFP-Pgm fusion in new developing MACs. (Panels a–f) A vegetative 51new cell was injected with linearized plasmid pPBL49-GFP-M, and autogamy was induced. MAC fragmentation was monitored by DAPI staining (panels a,c,e,g,i), and GFP fluorescence was examined with a Leica microscope (panels b,d,f,h,j). (White arrowheads) Developing new MACs. Note the punctuate nuclear GFP fluorescence pattern in panels d and f. Additional fluorescent speckles were sometimes observed with the GFP filter in noninjected cells (panels h,j) and in cells injected with GFP constructs (panel b), due to the presence of birefringent crystals in the cytoplasm. These do not colocalize with nuclear DAPI fluorescence.

Figure 2.

Post-transcriptional silencing of the PGM gene during MAC development. (A) Maps of genes PGM and PtSPO11 showing fragments used as Northern blot hybridization probes (gray line for PGM; the whole gene was used for PtSPO11), and for the construction of RNAi plasmids PGM-1 and PGM-2 (1 and 2 on PGM map) and PtSPO11-1 and PtSPO11-2 (1 and 2 on PtSPO11 map). The two PGM introns (thin lines) were confirmed by fine restriction analysis and sequencing of RT–PCR products. Restriction sites are indicated. (M) MscI; (H) HindIII; (N) NcoI; (Bs) BsmBI. (B) PGM silencing during conjugation. 51new mt7 and mt8 reactive cells were obtained in induced silencing bacteria harboring RNAi plasmids PGM-2 (P) or PtSPO11-1 (S). The silencing of ND7 (N) was used as a control RNAi. Following conjugation, mating pairs were transferred to each corresponding silencing medium. A conjugation in standard K. pneumoniae medium (K) was also performed. The phenotype of exconjugants is displayed as histograms (see footnote for Table 1). (C) PGM silencing following mating pair separation. Reactive cells were obtained in standard K. pneumoniae medium, and mating pairs were transferred to silencing medium.

Figure 3.

Inhibition of IES 51G4404 excision in PGM silenced 51ΔA autogamous cells. (A) Quantification of IES-retaining chromosomes (IES⁺) on Southern blots of PstI-restricted DNA during autogamy. (Gray box) Hybridization probe Gmac; (Vk) vegetative cells grown in K. pneumoniae before transfer to each silencing medium. All time points are in hours (see Supplemental Fig. S4). (B) LMPCR analysis of DSBs at the left arm of each IES excision site in the same time-course experiment.

Figure 4.

Inhibition of the imprecise deletion of the surface antigen A gene. A Southern blot of BglII–XhoI-restricted genomic DNA was hybridized with probe A (middle panels and histograms on the top), then with IES 51A2591 probe (bottom panels). Species 1 and 2 are absent from the vegetative old MAC (lanes Vk and V) and carry the full-length A gene (1: with IESs; 2: without IES, as in 51new). Species 3 is present in the old MAC of the particular clone used here and carries an internal deletion of the A gene. Species 4 is also found in the old MAC and carries a telomere branched upstream of A, as in the MAC of the d48 control (a macronuclear variant harboring a telomeric deletion of the A gene) (Forney and Blackburn 1988).