Because of their nuclear dimorphism, ciliates constitute unique unicellular models for the study of the molecular mechanisms involved in programmed DNA elimination during somatic differentiation. During development of the macronucleus (MAC) in Paramecium tetraurelia, ~30% of the germline DNA is eliminated from the somatic genome. Eliminated sequences include ~45,000 short, single-copy Internal Eliminated Sequences (IESs), which are excised through a highly precise “cut-and-close” mechanism related to DNA transposition. PiggyMac (Pgm), a catalytic active domesticated piggyBac transposase, is essential for the introduction of initiating DNA double-strand breaks at IES ends. The DNA double-strand break repair proteins Ku70/Ku80 interact with Pgm and are required for DNA cleavage at IES ends, ensures tight coupling between DNA cleavage and repair. Because IESs do not carry a clearly conserved sequence motif that could be recognized specifically by the excision machinery, one major issue is whether other partners are required to target Pgm to IES ends.
We recently discovered that nine additional Pgm-like (PgmL) proteins are encoded by the MAC genome. PGML genes group in five families of whole-genome duplication (WGD) ohnologs. In contrast to Pgm, PgmLs do not harbor a conspicuous DDD catalytic triad, suggesting that they may not have retained full catalytic activity. However, high-throughput RNA sequencing revealed that all PGML genes are transcribed specifically during MAC development, and fluorescence microscopy data indicated that all PgmL proteins localize in the developing new MAC. Systematic RNAi experiments, and a genome-wide survey of IES retention in depleted cells, established that each PgmL family is essential for IES excision. Using a heterologous protein expression system, we demonstrated that PgmLs can interact with each other and with Pgm in cell extracts. However, the nuclear localization of each PgmL family is differentially affected upon RNAi-mediated inactivation of other PGML genes. Our results indicate that Pgm acts within a highly ordered multi-component protein complex to catalyze programmed DNA elimination.