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.

Nuclear dualism without extensive DNA elimination in the ciliateLoxodes magnus

“…Secondary loss is more parsimonious, because karyorelicts are sister to heterotrichs, 42,66 in which at least one genus ( Blepharisma ) performs extensive genome editing like other ciliates. 9,12 The presence of Dcl genes in Loxodes , homologous to those involved in genome editing in other ciliates, also support secondary loss, whereas the apparent absence of a domesticated excisase is less conclusive, as ciliate excisases come from at least two different families, 12,19,21,67 and so were independently or repeatedly domesticated.…”

“…Introns were empirically annotated from RNA-seq mappings using the Intronarrator pipeline commit b6abd3b (https://github.com/Swart-lab/Intronarrator), because their short length made it difficult to model them effectively, as previously observed with Blepharisma . 12 Introns were identified from Hisat2 mappings of RNA-seq reads vs. the MAC and MIC Falcon assemblies with the following Intronarrator parameters: MIN_INTRON_RATIO=0.2, MIN_INTRONS=10, MAX_INTRON_LEN=40. Introns were then removed from the sequence to produce an artificial “intronless” assembly; non-coding RNAs were identified with Infernal v1.1.4 97 and hard-masked.…”

“…The same methods and thresholds were applied to call 6mA in MAC read data of Blepharisma stoltei . 12…”

How ciliates got their nuclei

“…But a few models cannot do justice to the richness of ciliate diversity (let alone protists). Ciliates include the majestic trumpet-shaped Stentor , able to regenerate itself from the tiniest cell fragment; Euplotes , which walks on leg-like bundles of coordinated cilia; Loxodes , which senses gravity using a subcellular organelle, not unlike a vertebrate inner ear; Didinium , which can paralyze and engulf prey far larger than itself in one “bite”; and Blepharisma , one of the few microorganisms that can easily be recognized without a microscope by its purple tinge, and the subject of the two papers in this issue from Seah, Singh, Swart, and colleagues ( 1 , 2 ).…”

“…Knowledge about Blepharisma therefore greatly clarifies our overall picture of ciliate evolution, in particular for ancient and highly variable traits like macronuclear differentiation and IES removal. And indeed, the authors describe a new category of IESs ( 1 ) and new mechanistic insights into the ancient origin of IES removal based on the PiggyBac transposase ( 2 ).…”