RNA-directed epigenetic regulation of DNA rearrangements

Abstract

Ciliated protozoa undergo extensive DNA rearrangements, including DNA elimination, chromosome breakage and DNA unscrambling, when the germline micronucleus produces the new macronucleus during sexual reproduction. It has long been known that many of these events are epigenetically controlled by DNA sequences of the parental macronuclear genome. Recent studies in some model ciliates have revealed that these epigenetic regulations are mediated by non-coding RNAs. DNA elimination in Paramecium and Tetrahymena is regulated by small RNAs that are produced and operated by an RNAi (RNA interference)-related mechanism. It has been proposed that the small RNAs from the micronuclear genome can be used to identify eliminated DNAs by whole-genome comparison of the parental macronucleus and the micronucleus. In contrast, DNA unscrambling in Oxytricha is guided by long non-coding RNAs that are produced from the somatic parental macronuclear genome. These RNAs are proposed to act as templates for the direct unscrambling events that occur in the developing macronucleus. The possible evolutionary benefits of these RNA-directed epigenetic regulations of DNA rearrangement in ciliates are discussed in the present chapter.

Figure 1. Life cycle and DNA rearrangements in ciliates

(A) Nuclear dimorphism and conjugation of Tetrahymena. Like most ciliates, Tetrahymena thermophila (picture) has two different types of nuclei (highlighted in red), a macronucleus (Mac) and a micronucleus (Mic), in a single cell. When enough nutrients are available, vegetative Tetrahymena grow by binary fission, and the macro- and micronucleus are divided independently. After prolonged starvation, two cells of complementary mating type fuse to start the sexual reproduction process, conjugation. Their micronuclei undergo meiosis, and one of the meiotic products survives and divides mitotically, giving rise to two gametic nuclei, one stationary and one migratory. Fertilization occurs after the migratory gametic nucleus crosses the conjugation bridge. The zygotic nucleus divides twice, and two products differentiate as macronuclei while the other two differentiate as micronuclei. The parental macronucleus becomes pyknotic and is resolved. (B) There are four types of DNA rearrangements in ciliates (see text for details). Green lines: macronuclear-destined sequences, red boxes: micronuclear-limited (eliminated) sequences, dashed green lines: telomeres. (C) Distribution of the four types of DNA rearrangements in ciliates. +: present, −: absent or not observed.

Figure 2. Parental macronuclear DNA sequences epigenetically regulate DNA rearrangement in the new macronucleus

Cells before conjugation (parent) are on the left and cells post-conjugation (progeny) are on the right. (A) DNA rearrangement at the A-gene locus in the wild type Paramecium strain. A Type II DNA rearrangement occurs, and a DNA segment (red) adjacent to the A-gene is eliminated during new macronuclear (Mac) development, while the A-gene is retained. (B) In the d48 mutant, the A-gene is absent in the parental Mac. While the micronuclear (Mic) A-gene locus is intact, the A-gene is eliminated from the new Mac. (C) Microinjection of DNA containing A-gene sequence into the d48 mutant results in the retention of the A-gene in the new Mac. (D) Type I DNA rearrangement in a wild type Paramecium strain. IESs are eliminated during new Mac development. (E) Microinjection of DNA containing the red IES results in the retention of this IES in the new Mac.

Figure 3. A model for small RNA-directed DNA rearrangement in Tetrahymena

In the early conjugation stages, the genome of the micronucleus (Mic), including the IESs, is transcribed bi-directionally, and these transcripts form dsRNA (a). The dsRNAs are processed into small RNAs (scnRNAs) by a Dicer-like RNase III (b), and the scnRNAs are transferred to the parental macronucleus (Mac) (c). In the mid conjugation stages, any scnRNAs homologous to DNAs in the parental Mac are degraded (d). In the late conjugation stages, the scnRNAs that are not degraded in the parental Mac (those homologous to IESs) are transferred to the developing new Mac (e), where they target IESs to be eliminated by base pairing (f).

Figure 4. A model for long RNA-guided DNA rearrangement in Oxytricha

(A) Telomere to telomere transcription of parental macronuclear “gene-sized” chromosomes produces guide RNAs (wavy line with circles), which are then transported to the newly developed macronucleus where they act as scaffolds to guide DNA rearrangements. This model explains the effect of disruption of guide RNAs by RNAi (B) and microinjection of artificial templates (C).