Transcriptome analysis of the binucleate ciliate Tetrahymena thermophila with asynchronous nuclear cell cycles

Abstract

As a prototypic ciliated protozoan, Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the 'cell cycles' of the diploid germline micronucleus and polyploid somatic macronucleus are offset. Micronuclear S phase initiates just before cell division and is completed in daughter cells prior to the onset of macronuclear S phase. Whereas mitotic micronuclear division occurs mid-cell cycle, amitotic macronuclear division immediately precedes cytokinesis. Here we report the first RNA-seq analysis across the cell cycle of a binucleated organism. RNA was isolated at 30 min intervals across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the predicted 26,000+ T. thermophila genes were shown to be cell cycle regulated. Proteins that are required in micro- and macronuclei exhibit a single mRNA peak that correlates with their macronuclear function, while the expression of nucleus-limited protein-coding genes, including nucleoporins and importins, peak prior to their respective nucleus-specific role. Cyclin D and cyclin A/B genes showed distinct expression patterns that predict nucleus-specific functions. Clustering of periodically expressed genes revealed seven gene expression patterns. Four clusters have known PANTHER GO biological processes that are overrepresented for G1/S and G2/M phase functions. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication and cell division.

Developmentally programmed switches in DNA replication: gene amplification and genome-wide endoreplication in Tetrahymena

“…The 2 h Gap-S-Gap-S endocycle in starved mating populations was asynchronous, as anticipated based on differences in the timing for mating pair formation. By comparison, macronuclear S phase constitutes about 1/3 of the ~3 h vegetative cell cycle (Zhang et al, 2022).…”

“…Could these hypothetical structures serve as molecular beacons for the DNA replication machinery when ORC is rate-limiting? For those interested in pursuing gene expression, preliminary raw and processed RNAseq datafiles on endoreplication phases I and II cells were previously deposited to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE210103, along with cell cycle analysis of the vegetative cell cycle (Zhang et al, 2022). TXR1 knockout mutant (generated by macronuclear gene disruption and phenotypic assortment) (Gao et al, 2013).…”

“…For those interested in pursuing gene expression, preliminary raw and processed RNA-seq datafiles on endoreplication phases I and II cells were previously deposited to the NCBI Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE210103, along with cell cycle analysis of the vegetative cell cycle (Zhang et al, 2022).…”

“…ORC-dependent DNA replication is coordinately regulated in vegetative growing Tetrahymena, such that the heterochromatic micronucleus and euchromatic macronucleus replicate their chromosomes at non-overlapping stages of the cell cycle (reviewed in Cole and Sugai, 2012;Zhang et al, 2022). Development is much more complex: multiple rounds of micro-and macronuclear DNA replication, meiosis and mitosis occur in parental and post-zygotic progeny.…”

“…Like all ciliated protozoa, T. thermophila harbors two functionally distinct nuclei within its cytoplasm: the transcriptionally silent, diploid ‘germline’ micronucleus and the transcriptionally active, polyploid ‘somatic’ macronucleus (reviewed in Karrer, 2012). ORC-dependent DNA replication is coordinately regulated in vegetative growing Tetrahymena, such that the heterochromatic micronucleus and euchromatic macronucleus replicate their chromosomes at non-overlapping stages of the cell cycle (reviewed in Cole and Sugai, 2012; Zhang et al, 2022). Development is much more complex: multiple rounds of micro- and macronuclear DNA replication, meiosis and mitosis occur in parental and post-zygotic progeny.…”