Small RNA-directed DNA elimination: the molecular mechanism and its potential for genome editing

Figures & data

Table 1. Proteins involved in DNA elimination in Tetrahymena

Protein	Description	Reference
Coi6p	Boundary-protecting factor	Suhren et al. [21]
Coi7p	Boundary-protecting factor	Suhren et al. [21]
Dcl1p	Dicer-like 1 protein, processes scnRNAs	Malone et al. [7]
Ema1p	RNA helicase, unwinds nascent RNAs transcribed from IESs	Aronica et al. [19]
Ezl1p	Histone methyltransferase, catalyses H3K27 methylation at IESs	Liu et al. [16]
Giw1p	Regulates nuclear localization of Twi1p	Noto et al. [18]
Jmj1p	Boundary-protecting factor	Suhren et al. [21]
Lia5p	Boundary-protecting factor	Suhren et al. [21]
Pdd1p	Chromodomain protein, binds H3K27me3 and H3K9me3	Liu et al. [16]
TKu80p	Ku80 homologue, protects the ends of double-strand breaks created by Tpb2p	Lin et al. [23]
Tpb1p	PiggyBac-like transposase, excises a small subset of IESs	Cheng et al. [9]
Tpb2p	PiggyBac-like transposase, excises the majority of IESs	Cheng et al. [13]
Tpb6p	PiggyBac-like transposase, excises a small subset of IESs	Cheng et al. [9]
Twi1	Piwi protein, uses a scnRNA to locate IESs	Mochizuki et al. [2]
Twi11	Piwi protein, uses a scnRNA to locate IESs	Mochizuki et al. [2]

Figure 1. Tetrahymena life cycle. Each cell contains a macronucleus (MAC) and a micronucleus (MIC). In the presence of sufficient nutrients, Tetrahymena reproduces asexually by binary fission (A). However, when there is a lack of nutrients, it reproduces sexually by conjugation (B-J). To start conjugation, two cells of complementary mating types fuse (B) and their MICs undergo meiosis (C). Three of the meiotic products are degraded, while the surviving nucleus undergoes mitosis (D). The fused cells then exchange a pronucleus (E) and the pronuclei fuse to create a zygotic nucleus (F), which undergoes two rounds of mitosis (G). Two products will develop into new MACs, one will form a new MIC and the fourth product is degraded. The parental MAC is also degraded and the fused cells separate (H). Finally, the MIC divides mitotically (I), which is followed by binary fission (J). The approximate time-scale of events is indicated in hours post-mixing (hpm)

Figure 2. Programmed DNA elimination. Early scan-RNAs (scnRNAs) are produced from type-A internal eliminated sequences (IESs) and surrounding regions (A). Next, they are transported to the MAC (B) and scnRNAs complementary to the MAC genome are degraded. Meanwhile, a new MIC and MAC have been formed (C). The remaining scnRNAs are transported to the new MAC, where they recognize both type-A and – B IESs (D). The scnRNAs induce heterochromatin formation and the production of late-scnRNAs, which further spread the heterochromatin (E). Finally, the parts of the genome marked by heterochromatin are excised and the ends are ligated (F)

Figure 3. From Twi1p-scnRNA target search to IES elimination. Together with Ema1p, the Twi1p-scnRNA complex searches for targets in nascent transcripts (A). At matching sequences, histone H3 is methylated at lysine 9 (H3K9me3) and 27 (H3K27me3) by Ezl1p (B). Pdd1p is recruited to the heterochromatin (C). This is a requirement for the production of late scnRNAs that further spread the heterochromatin. At the same time, boundary-protecting factors confine the heterochromatin to IESs (D). Finally, Tpb2p excises IESs marked by heterochromatin (E)

Suhren JH, Noto T, Kataoka K, et al. Negative regulators of an RNAi-heterochromatin positive feedback loop safeguard somatic genome integrity in tetrahymena. Cell Rep. 2017 Mar;18(10):2494–2507.

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Liu Y, Taverna SD, Muratore TL, et al. RNAi-dependent H3K27 methylation is required for heterochromatin formation and DNA elimination in Tetrahymena. Genes Dev. 2007 Jun;21(12):1530–1545.

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Noto T, Kurth HM, Kataoka K, et al. The tetrahymena argonaute-binding protein giw1p directs a mature argonaute-siRNA complex to the nucleus. Cell. 2010 Mar;140(5):692–703.

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Cheng CY, Young JM, Lin CYG, et al. The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila. Genes Dev. 2016 Dec;30(24):2724–2736.

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