Genome editing excisase origins illuminated by somatic genome of Blepharisma

Abstract

The germ-soma distinction is a defining feature of multicellular eukaryotes. Analogous to this, ciliates, a ubiquitous microbial eukaryote lineage, have morphologically and functionally distinct nuclei, but within single cells: the germline micronucleus (MIC) and somatic macronucleus (MAC). The origins and mechanisms of the MIC to MAC transformation, especially the extensive elimination of abundant internally eliminated sequences (IESs) and transposons during genome reorganization, are great biological mysteries. Blepharisma represents one of the two earliest diverging ciliate classes, and has unique, dual pathways of MAC development, making it ideal for investigating the functioning, origins and evolution of these processes. Here, we report the MAC genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mb), arranged as numerous alternative telomere-capped minichromosomes, tens to hundreds of kilobases long. The B. stoltei MAC genome encodes eight PiggyBac transposase homologs liberated from transposons. All are subject to purifying selection, but just one, the putative Blepharisma IES excisase, has a complete catalytic amino acid triad. Numerous genes encoding other domesticated transposases are present in B. stoltei, and often are comparably strongly upregulated in a similar timeframe to model ciliate genome reorganization homologs. Our phylogenetic investigations suggest the PiggyBac homologs may have been ancestral ciliate IES excisases. The B. stoltei MAC genome, together with the upcoming MIC genome, highlights the evolution and complex interplay between transposons, domesticated transposases, and genome reorganization in the context of germline-soma differentiation within single cells.

MITE infestation accommodated by genome editing in the germline genome of the ciliate Blepharisma

“…To investigate the Blepharisma stoltei (hereafter Blepharisma ) germline genome, we enriched germline MICs from strain ATCC 30299 and reconstructed 39,799 IESs (13.2 Mbp total, average coverage ~45×) scaffolded on the previously assembled 41 Mbp somatic genome ( 37 ). We applied a mapping and targeted assembly approach developed for PacBio long reads ( 39 ), which could better assemble repetitive elements compared to using short read sequencing ( SI Appendix , SI Results “IES assembly from short vs. long reads” ).…”

“…S7 B and SI Results “Diversity of MAC-limited non-LTR retrotransposon-derived repeats” ). All of Blepharisma ’s MAC genome-encoded transposases appear domesticated (i.e., have no TIRs) ( 37 ), and none have replicated to the same extent as the retrotransposon-derived repeats in this genome. Unlike the Bogo and BstTc1-derived elements, more retrotransposon-derived sequences, containing the reverse transcriptase domain RVT_1, were detected in the Blepharisma MAC genome than in assembled IESs ( Fig.…”

“…Twenty-nine genes in the main somatic assembly encoded full or partial copies of reverse transcriptase domain RVT_1 ( 37 ). The four longest retrotransposon genes also encoded an N-terminal apurinic/apyrimidinic endonuclease (Exo_endo_phos_2) domain upstream of RVT_1.…”

“…Blepharisma has been a laboratory model for photobiology ( 32 ) and for mating type recognition through diffusible mating factors (gamones) ( 33 – 36 ), so cultivated strains and protocols for inducing conjugation and development are available. An accurate, highly contiguous draft sequence of the somatic genome is now also available ( 37 ). The somatic genome encodes a probable IES excisase, Blepharisma PiggyMac (BPgm), a PiggyBac-family homolog that is most closely related to the main IES excisases of Paramecium (PiggyMac) and Tetrahymena (Tpb2).…”

“…Other somatic PiggyBac paralogs are also present but lack the complete “catalytic triad” of the classical PiggyBac transposase from cabbage looper moths that is necessary for excisase activity, similar to the situation in Paramecium ( 38 ). BPgm is upregulated during the formation of the new MAC together with other development-specific genes, including homologs of sRNA biogenesis proteins implicated in genome editing ( 37 ).…”