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

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.

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

During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of Blepharisma stoltei, a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.

Comparative genome analysis of three euplotid protists provides insights into the evolution of nanochromosomes in unicellular eukaryotic organisms.

One of the most diverse clades of ciliated protozoa, the class Spirotrichea, displays a series of unique characters in terms of eukaryotic macronuclear (MAC) genome, including high fragmentation that produces nanochromosomes. However, the genomic diversity and evolution of nanochromosomes and gene families for spirotrich MAC genomes are poorly understood. In this study, we assemble the MAC genome of a representative euplotid (a new model organism in Spirotrichea) species, Euplotes aediculatus. Our results indicate that: (a) the MAC genome includes 35,465 contigs with a total length of 97.3 Mb and a contig N50 of 3.4 kb, and contains 13,145 complete nanochromosomes and 43,194 predicted genes, with the majority of these nanochromosomes containing tiny introns and harboring only one gene; (b) genomic comparisons between E. aediculatus and other reported spirotrichs indicate that average GC content and genome fragmentation levels exhibit interspecific variation, and chromosome breaking sites (CBSs) might be lost during evolution, resulting in the increase of multi-gene nanochromosome; (c) gene families associated with chitin metabolism and FoxO signaling pathway are expanded in E. aediculatus, suggesting their potential roles in environment adaptation and survival strategies of E. aediculatus; and (d) a programmed ribosomal frameshift (PRF) with a conservative motif 5'-AAATAR-3' tends to occur in longer genes with more exons, and PRF genes play an important role in many cellular regulation processes. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00175-0.

Comparative Genome Analysis Reveals Cis-Regulatory Elements on Gene-Sized Chromosomes of Ciliated Protists.

Gene-sized chromosomes are a distinct feature of the macronuclear genome in ciliated protists known as spirotrichs. These nanochromosomes are often only several kilobase pairs long and contain a coding region for a single gene. However, the ways in which transcription is regulated on nanochromosomes is still largely unknown. Here, we generated macronuclear genome assemblies for two species of Pseudokeronopsis ciliates to better understand transcription regulation on gene-sized chromosomes. We searched within the short subtelomeric regions for potential cis-regulatory elements and identified distinct AT-rich sequences conserved in both species, at both the 5' and 3' end of each gene. We further acquired transcriptomic data for these species, which showed the 5' cis-regulatory element is associated with active gene expression. Gene family evolution analysis suggests nanochromosomes in spirotrichs may originated approximately 900 million years ago. Together our comparative genomic analyses reveal novel insights into the biological roles of cis-regulatory elements on gene-sized chromosomes.