Origins of genome-editing excisases as illuminated by the somatic genome of the ciliate Blepharisma

Abstract

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout the ciliate germline genome are deleted during the development of the streamlined somatic genome. The genus Blepharisma represents one of the two high-level ciliate clades (subphylum Postciliodesmatophora) and, unusually, has dual pathways of somatic nuclear and genome development. This makes it ideal for investigating the functioning and evolution of these processes. Here we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mbp), arranged as numerous telomere-capped minichromosomal isoforms. This genome encodes eight PiggyBac transposase homologs no longer harbored by transposons. All appear subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We hypothesize that PiggyBac homologs were ancestral excisases that enabled the evolution of extensive natural genome editing.

Keywords: PiggyBac; PiggyMac; natural genome editing; transposase; transposon.

Fig. 1.

Blepharisma nuclei and nuclear development during conjugation. (A) Cell of B. stoltei strain ATCC 30299 stained with anti-alpha-tubulin-Alexa488 (depth color-coded) and the dsDNA dye DAPI (cyan). (B) Snapshot of a 3D reconstruction (Imaris, Bitplane) from CLSM fluorescence images of a cell stained with the dsDNA dye Hoechst 33342 (Invitrogen). (C) Schematic of the nuclear processes occurring during conjugation in Blepharisma, classified according to, and modified from figure 45 of ref. (copyright, Elsevier). During conjugation, half of the MICs in each cell undergo meiosis (meiotic MICs), and the rest do not (somatic MICs). One of the meiotic MICs eventually gives rise to two haploid gametic nuclei, one of which (the migratory nucleus) is exchanged with that of its partner. Subsequently, the migratory and stationary haploid nuclei fuse to generate a zygotic nucleus (synkaryon), which, after successive mitotic divisions, gives rise to both new MICs and new MACs (known as primary anlagen). The new MACs continue to mature, eventually growing in size and DNA content (6). In parallel, secondary macronuclear anlagen develops directly, and with time, the old MAC condenses and degrades. After karyogamy, cells are classified into ten stages: S (synkaryon), D1 (first mitosis), I1 (first interphase), D2 (second mitosis), I2 (second interphase), D3 (third mitosis), I3 (third interphase), D4 (fourth mitosis), E1 (first embryonic stage), and E2 (second embryonic stage; not shown).

Fig. 2.

Comparison of basic properties of ciliate MAC genomes. In cell diagrams, MACs are green and MICs are small black dots in close proximity to MACs. Citations for genome properties are in Dataset S1.

Fig. 3.

Gene-dense somatic genome. HiFi (DNA) and RNA-seq coverage across a representative B. stoltei ATCC 30299 MAC genome contig (Contig_1). Y scale is linear for HiFi reads and logarithmic (base 10) for RNA-seq. Plus strand (relative to the contig) RNA-seq coverage is green; minus strand RNA-seq coverage is blue. Between the RNA-seq coverage graphs, each horizontal arrow represents a predicted gene. Two orthogroups classified by OrthoFinder are shown.

Fig. 4.

Developmental staging of B. stoltei for RNA-seq. Classification of nuclear morphology into stages is according to previous descriptions (6). Nuclear events occurring before and up to, but not including fusion of the gametic nuclei (syngamy) are classified into sixteen stages indicated by roman numerals. These are the pre-gamic stages of conjugation where the MICs undergo meiosis and the haploid products of meiotic MICs are exchanged between the conjugating cells. Stages after syngamy are classified into ten stages as in Fig. 1. Illustration of various cell stages (adapted from ref. 39). Stacked bars show the proportion of cells at each time point at different stages of development, preceded by the number of cells inspected (n).

Fig. 5.

MAC genome-encoded transposases in ciliates and properties of a putative Blepharisma IES excisase. (A) Presence/absence matrix of PFAM transposase domains detected in predicted MAC genome-encoded ciliate proteins. Ciliate classes are indicated before the binomial species names. (B) DDE_Tnp_1_7 domain phylogeny with PFAM domain architecture and gene expression heatmap for Blepharisma. “Mixing” indicates when cells of the two complementary mating types were mixed. Outgroup: PiggyBac element from Trichoplusia ni. Catalytic residues: D—aspartate, D'—aspartate residue with 1 aa translocation.

Fig. 6.

Phylogeny of ciliate PiggyBac homologs and eukaryotic PBLEs. The highlighted clade contains all PiggyBac homologs found in Heterotrichea, containing MAC and MIC-limited homologs of PiggyMac from Blepharisma and PiggyMac homologs of C. magnum. The tree is rooted at the PiggyBac-like element of Entamoeba invadens.