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DNA rearrangements directed by non‐coding RNAs in ciliates

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Extensive programmed rearrangement of DNA, including DNA elimination, chromosome fragmentation, and DNA unscrambling, takes place in the newly developed macronucleus during the sexual reproduction of ciliated protozoa. Recent studies have revealed that two distant classes of ciliates use distinct types of non‐coding RNAs to regulate such DNA rearrangement events. DNA elimination in Tetrahymena is regulated by small non‐coding RNAs that are produced and utilized in an RNA interference (RNAi)‐related process. It has been proposed that the small RNAs produced from the micronuclear genome are used to identify eliminated DNA sequences by whole‐genome comparison between the parental macronucleus and the micronucleus. In contrast, DNA unscrambling in Oxytricha is guided by long non‐coding RNAs that are produced from the parental macronuclear genome. These long RNAs are proposed to act as templates for the direct unscrambling events that occur in the developing macronucleus. Both cases provide useful examples to study epigenetic chromatin regulation by non‐coding RNAs. Copyright © 2010 John Wiley & Sons, Ltd.

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Figure 1.

Nuclear dimorphism in Tetrahymena. Like most ciliates, Tetrahymena thermophila has two different types of nuclei (highlighted in red) in a single cell: a macronucleus (Mac) and a micronucleus (Mic). The micronucleus has 5 chromosomes per haploid genome, whereas the macronucleus has over 20,000 chromosomes.

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Figure 2.

Life cycle of Tetrahymena. When enough nutrients are available, vegetative Tetrahymena cells multiply by binary fission and the macronucleus and micronucleus divide independently (a). After prolonged starvation, two cells of complementary mating types fuse to start the sexual reproduction process of conjugation (b). Their micronuclei undergo meiosis (c) and one of the meiotic products survives and divides mitotically, giving rise to two gametic nuclei, one of which is stationary and the other migratory (d). The migratory gametic nucleus crosses the conjugation bridge (e) and the gametic nuclei fuse to produce a diploid zygotic nucleus (f). The zygotic nucleus undergoes two mitotic divisions (g), and two of the products differentiate as macronuclei, whereas the other two differentiate as micronuclei (h). The parental macronucleus becomes pyknotic and is resorbed. The pairing dissolves and one of the two micronuclei is degraded (i). Finally, when nutrients are available again, the progeny resumes vegetative growth by cytokinesis without macronuclear (but with micronuclear) division (j).

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Figure 3.

DNA rearrangement and endoreplication in ciliates. (a) DNA elimination and chromosome fragmentation in Tetrahymena. Numerous internal eliminated sequences (IESs, green lines marked with the letter ‘i’) are removed and two flanking macronuclear‐destined sequences (MDSs) are re‐ligated. In parallel, chromosome breakage at the Cbs (chromosome breakage sequence, marked with an arrow and the letter ‘c’) occurs and new telomeres (red triangles) are formed. The macronuclear chromosomes are eventually endoreplicated to around 50 copies. (b) rDNA rearrangement in Tetrahymena. The single micronuclear rDNA locus (blue arrow) is excised and rearranged into an inverted repeat. Telomeres are formed at both ends de novo and endoreplicated to approximately 10,000 copies. (c) DNA unscrambling in spirotrich ciliates (e.g., Oxytricha). Many genes are fragmented, ‘scrambled’ (that is, not 1‐2‐3‐4‐5‐6 but 1‐3‐2‐4‐5‐6, in this example) and some segments are inverted (segment 5 in this example) and separated by IESs (green lines marked with ‘i’) in the micronucleus (Mic). They are joined and assembled (unscrambled) into the proper order and direction in the macronucleus (Mac)..

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Figure 4.

Elimination of transposon repeats from the macronucleus. Tetrahymena cells in a vegetative state were used to detect two distinct transposon‐related sequences, Tlr1 (top) and REP (bottom), by fluorescent in situ hybridization (FISH, green, left). DNA was stained with DAPI (magenta, middle). The micronucleus (i) and the macronucleus (a) are marked.

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Figure 5.

A model for small RNA‐directed DNA rearrangement in Tetrahymena. In the early conjugation stages, the genome of the micronucleus (Mic), including the internal eliminated sequences (IESs), is transcribed bi‐directionally (a) and the resulting transcripts form double‐stranded RNAs (dsRNA) molecules (b). The dsRNAs are processed into small RNAs (scnRNAs) (c). The scnRNAs are transferred to the parental macronucleus (Mac) and any scnRNAs homologous to DNA sequences in the parental Mac are degraded in the mid‐conjugation stages (d). In late conjugation stages, the scnRNAs that were 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).

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Figure 6.

Small RNA‐directed DNA rearrangement in Tetrahymena. (a) Promiscuous bi‐directional transcription of the micronuclear genome by RNA polymerase II (RNAPII) produces double‐stranded micronuclear non‐coding (nc) RNA. (b) The Dicer‐like protein Dcl1p digest long double‐stranded micronuclear ncRNA to short (∼28–29 nt) RNAs, named scnRNAs, in the micronucleus (Mic). (c) scnRNA makes complex with the Argonaute protein Twi1p and the endoribonuclease (Slicer) activity of Twi1p cuts passenger strand of scnRNA. Giw1p binds to Twi1p complexed with single‐stranded scnRNA and transports the complex to parental macronucleus (Mac). (d) The RNA methyltransferase Hen1p 2‐O‐methylates single‐stranded scnRNAs most likely in the parental macronucleus. This modification stabilizes scnRNA. (e, f) The RNA helicase Ema1p facilitates interaction between Twi1p–scnRNA complex and nascent macronuclear ncRNA. This interaction induces scnRNA degradation in the parental macronucleus and recruits the histone methyltransferase Ezl1p in the new macronucleus. (g) Ezl1p catalyzes methylations of histone H3 at lys9 and lys27. (h) The chromodomain proteins Pdd1p and Pdd3p bind to the methylated histone H3 and establish heterochromatin‐like structure. (i) The PiggyBac transposase‐like protein Tpb2p, which has endonucleotidase activity and is required for DNA elimination, is likely involved in the final DNA excision process. Events occurring sequentially are shown from top to bottom. The approximate stages at which the events occur are indicated on the right by arrows.

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Figure 7.

Long non‐coding RNA‐guided DNA unscrambling in Oxytricha. (a) A model for RNA‐guided DNA unscrambling in Oxytricha. Telomere‐to‐telomere transcription of parental macronuclear ‘gene‐sized’ chromosomes produces guide RNAs (wavy lines with circles), which are then transported to the newly developed macronucleus where they act as scaffolds to guide DNA rearrangement. (b) Disruption of long non‐coding RNAs by RNAi causes a defect in DNA rearrangement. (c) Microinjection of artificial templates (in this example, RNA having a 1‐2‐4‐3‐5‐6 sequence) alters the order of the DNA unscrambling pattern in the new macronucleus. (d) Microinjection of artificial templates that have base substitutions (C to U in this example) alters the DNA sequence of the new macronucleus.

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