UC Santa Cruz

Oxytricha trifallax, like all ciliated protozoans, possess two distinct types of nuclei: a germline micronucleus that is transcriptionally silent and a somatic macronucleus used for vegetative growth of the cells that is transcriptionally active. While the micronucleus resembles a typical eukaryotic nucleus with DNA organized on long chromosomes, micronuclear genes are interrupted by nongenic DNA and often exist in a non-linear, scrambled order. After ciliate mating, the parental macronucleus provides genetic information for the formation of a new macronucleus, which is derived from a newly formed diploid, micronuclear precursor. During this micronucleus to macronucleus transition, the micronuclear genome is modified drastically through various processing events to yield small, gene-sized molecules called nanochromosomes. During the multi-stage macronuclear development process, the near 1 Gb micronuclear genome is reduced to roughly 5% of its original DNA sequence complexity, with the resulting macronuclear nanochromosomes varying greatly in copy number. In addition to excision and removal of nongenic DNA and transposable elements (TEs), the developing macronucleus must sort and reorder the remaining coding segments into functional genic open reading frames for healthy vegetative growth of the cells. Here, I investigate the potential roles of small RNAs (sRNAs) in these macronuclear development processes, with an emphasis on a class called 27macRNAs, highly expressed after Oxytricha mating, during the early stages of macronuclear development. In addition, to explore the genes required for this complex macronuclear development process, I performed next generation sequencing of Oxytricha mRNAs from vegetative cells and from various timepoints during ciliate mating and macronuclear development. We have identified at least 5 regulatory groups or modules consisting of genes whose expression is highly co-regulated during the distinct stages of this process. We find that a disproportionate number of the mRNAs upregulated during this process encode for proteins involved in DNA and RNA metabolism, with the majority of these genes encoding evolutionarily conserved proteins across species.