The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila

Abstract

Ciliated protozoans perform extreme forms of programmed somatic DNA rearrangement during development. The model ciliate Tetrahymena thermophila removes 34% of its germline micronuclear genome from somatic macronuclei by excising thousands of internal eliminated sequences (IESs), a process that shares features with transposon excision. Indeed, piggyBac transposon-derived genes are necessary for genome-wide IES excision in both Tetrahymena (TPB2 [Tetrahymena piggyBac-like 2] and LIA5) and Paramecium tetraurelia (PiggyMac). T. thermophila has at least three other piggyBac-derived genes: TPB1, TPB6, and TPB7. Here, we show that TPB1 and TPB6 excise a small, distinct set of 12 unusual IESs that disrupt exons. TPB1-deficient cells complete mating, but their progeny exhibit slow growth, giant vacuoles, and osmotic shock sensitivity due to retention of an IES in the vacuolar gene DOP1 (Dopey domain-containing protein). Unlike most IESs, TPB1-dependent IESs have piggyBac-like terminal inverted motifs that are necessary for excision. Transposon-like excision mediated by TPB1 and TPB6 provides direct evidence for a transposon origin of not only IES excision machinery but also IESs themselves. Our study highlights a division of labor among ciliate piggyBac-derived genes, which carry out mutually exclusive categories of excision events mediated by either transposon-like features or RNA-directed heterochromatin.

Comprehensive Chromosome End Remodeling During Programmed DNA Elimination

“…Domesticated transposases, such as RAG1-RAG2 for V(D)J recombination [60][61][62] and PiggyBac in ciliate DNA elimination [63][64][65][66], recognize specific sequences in the genome during development to generate DSBs. However, we have not identified any transposases or their signatures in Ascaris that might generate the DNA breaks.…”

Transposable Element Domestication As an Adaptation to Evolutionary Conflicts

“…In Tetrahymena , IES are primarily intergenic and while TPB2 has retained catalytic activities that are essential for IES removal [60], LIA5 appears to have lost its catalytic activity but remain essential for DNA elimination likely through its involvement in chromatin reorganization prior to IES excision [57, 58]. Finally, TPB1 and TPB6 appear to function as catalytically active transposases, but differ from TPB2 in being dedicated to the removal of a small subset of IES that resemble ancient piggyBac transposons [59]. …”

“…In Paramecium , a fraction of IES appears to be derived from recent Tc1/ mariner -like element invasions and resemble miniature elements or solitary TIRs, whose sequences appear to have converged to be excised by Paramecium’s PiggyMac leaving no scar behind unlike typical Tc1/ mariner transposition [61]. In Tetrahymena , there is also substantial overlap and terminal sequences similarities between IES and TEs, including piggyBac -like elements [53, 59, 62]. These observations and what is known about the biochemistry of IES excision [61] support the idea that the process of IES elimination in Paramecium and Tetrahymena closely resembles the excision of piggyBac transposons, and that this type of elements could have provided both the enzymatic machinery and at least some of the cis-acting sequences now required for the process.…”

Evolutionary origins and impacts of genome architecture in ciliates

“…While TEs are tightly linked with the evolution of ciliate germ‐line genome architecture, they have also become an indispensable player in programmed DNA elimination. In Paramecium tetraurelia and T. thermophila , domesticated PiggyBac transposases (e.g., PiggyMAC or TPB encoded in the Paramecium and Tetrahymena somatic genomes, respectively) perform the bulk excision of germline‐limited DNA, including TEs, from the developing somatic genome . Silencing P. tetraurelia ’s PiggyMac transposase during development ultimately results in the retention of most of the ∼45,000 IESs, resulting in a nonfunctional somatic genome .…”

“…In Paramecium tetraurelia and T. thermophila, domesticated Piggy-Bac transposases (e.g., PiggyMAC or TPB encoded in the Paramecium and Tetrahymena somatic genomes, respectively) perform the bulk excision of germline-limited DNA, including TEs, from the developing somatic genome. [35][36][37][38][39] Silencing P. tetraurelia's PiggyMac transposase during development ultimately results in the retention of most of the ∼45,000 IESs, resulting in a nonfunctional somatic genome. 25 Although taming transposons appears to be required for the massive DNA elimination observed in ciliates, the degree of domestication (i.e., recruitment into the somatic genome versus limited to the germline) is variable.…”