RNA-Mediated Epigenetic Programming of Genome Rearrangements

Abstract

RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa. Maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transport somatic mutations to the next generation. This opportunity for RNA-mediated genome rearrangement and DNA repair is profound in the ciliate Oxytricha, which deletes 95% of its germline genome during development in a process that severely fragments its chromosomes and then sorts and reorders the hundreds of thousands of pieces remaining. Oxytricha's somatic nuclear genome is therefore an epigenome formed through RNA templates and signals arising from the previous generation. Furthermore, this mechanism of RNA-mediated epigenetic inheritance can function across multiple generations, and the discovery of maternal template RNA molecules has revealed new biological roles for RNA and has hinted at the power of RNA molecules to sculpt genomic information in cells.

Figure 1

An example of the ciliate sexual cycle. (a) A vegetative Paramecium cell. (b) Meiosis of two micronuclei. The macronucleus begins its fragmentation. (c) Seven out of eight haploid meiotic products degenerate; one (shown at top) divides mitotically, producing two identical gametic nuclei. During conjugation, one of the gametic nuclei is exchanged between partner cells. In the case of autogamy (self-fertilization), the two identical gametic nuclei fuse together. (d ) A cell that contains the zygotic nucleus. (e) The zygotic nucleus undergoes two subsequent mitotic divisions. (f) Two of the products differentiate into new macronuclei. ( g) Two karyonidal cells are present; each contains one new macronucleus, two micronuclei, and fragments of the old macronucleus.

Figure 2

Types of genome rearrangements during macronuclear development in ciliates. The micronuclear genome undergoes amplification to varying levels between and sometimes within different species, and three types of DNA elimination can occur (not necessarily in the order shown). One is imprecise elimination of germline-specific repeats, including all transposons and minisatellites. This step is often associated with chromosome fragmentation and healing of chromosome ends by de novo telomere addition (red ). The second type of rearrangement is the precise excision of internal eliminated sequences (IESs, yellow), which occurs in both Paramecium and Oxytricha. Stichotrichs, such as Oxytricha, also have a third layer of descrambling segment order and orientation. Precise DNA elimination and/or unscrambling restores the coding regions to construct functional open reading frames (thick black arrow). Abbreviations: MIC, micronucleus; MAC, macronucleus.

Figure 3

Mendelian and non-Mendelian inheritance in Paramecium. (a) Mendelian pattern of allelic segregation in a cross between A/A and a/a homozygotes. Conjugation produces two genetically identical heterozygotes, A/a. Autogamy of the heterozygous clones results in entirely homozygous clones with a 50% chance of receiving each of the parental genotypes. (b) Non-Mendelian maternal inheritance of mating types in P. tetraurelia. Each mating type (O or E) follows a cytoplasmic pattern of inheritance that is determined by the macronuclei. Mating type is programmed at every sexual reproduction by the maternal macronucleus via the maternal cytoplasm. Figure based on experiments in Reference .

Figure 4

Epigenetic inhibition of internal eliminated sequence (IES) excision. (a) In wild-type cells, all IESs and transposons (orange) are removed during development of the new macronucleus (MAC). Tiny red lines indicate telomeres at MAC chromosome ends. During sexual reproduction, the parental MAC is lost, and the new MAC develops from the parental micronucleus (MIC). (b) Microinjection of the MAC with an IES sequence (short orange bar matching the genomic IES sequence on the right) specifically blocks excision of this IES in the MAC of sexual progeny (26).

Figure 5

The scan RNA model for programmed genome rearrangements. (a) Endogenous short RNAs (scnRNAs, blue and orange dashed lines) are produced in the meiotic micronucleus (MIC). (b) These scnRNAs are exported to the maternal macronucleus (MAC), where they may scan the genome via interactions with long maternal transcripts (blue wavy lines). The scnRNAs at this point correspond to the entire germline genome, including transposons and internal eliminated sequences (IESs, both in orange). (c) scnRNAs (orange dashed lines) that cannot pair with homologous sequences are then selectively transported to the developing (zygotic) MAC, where they target histone methylation on homologous DNA sequences. (d ) The process in panel c results in the specific elimination of micronuclear sequences that are absent from the maternal MAC. Tiny red lines indicate telomeres.

Figure 6

A template model for RNA-guided genome rearrangements in Oxytricha. (a) Bidirectional maternal template transcripts ( green wavy line) are produced from short macronuclear chromosomes and then transported to the developing macronucleus (MAC). (b) Here, the RNA transcripts act as scaffolds to guide rearrangements (deletion, permutation, and inversion) of corresponding micronuclear DNA sequences. (c) This process leads to the complete elimination of micronucleus (MIC)–specific DNA (orange boxes). (d ) De novo telomere addition (red boxes) and amplification complete the formation of new macronuclear chromosomes.

Figure 7

RNA-mediated epigenetic reprogramming of a DNA inversion in Oxytricha. This example illustrates a new scrambled pattern in the nonscrambled gene encoding telomere end–binding protein subunit β (TEBPβ) that arises from the introduction of a synthetic RNA template containing an inversion. (a) To create the artificial template, macronucleus-destined sequence (MDS) junctions flanking target segment 4 were slightly adjusted to program two new recombination breakpoints, accommodating short existing repeats as new pointers (TGGT between the 3′ end of segment 3 and the 3′ end of segment 4, and CTTC to fuse the 5′ end of segment 4 to the 5′ end of segment 5; underlined sequences are embedded in the wild-type ACTTC pointer between MDS 4 and 5; the TGGT breakpoint begins 5 bp upstream of the wild-type ACTC pointer between MDS 3 and 4). The resulting alternative, 1.8-kb template containing inverted segment 4 (inv4) is 27 nt shorter than wild-type TEBPβ. Two independently microinjected constructs, inv4⁺ and inv4²⁺, contain different numbers of substitutions within 36 nt of the inverted junctions to distinguish injected molecules from endogenous, rearranged progeny DNA and to query substitution transfer. (b) Genomic DNA extracted from the F₁ progeny of cells injected with either sense (s) or antisense (as) template transcripts were amplified by polymerase chain reaction (as in 103) through the use of the primers indicated by arrows in panel a. The 231-bp product contains the inversion (inv) relative to the 258-bp wild-type (WT) product in noninjected control cells (ctrl). M indicates the 1-kb plus DNA ladder (Invitrogen, Carlsbad, California). This example illustrates that efficient epigenetic reprogramming of the DNA-rearrangement pathway occurs in this gene, although the WT version is also present. DNA sequencing confirmed that the 231-bp products contain endogenous, rearranged molecules with the programmed DNA inversion. Sequences are provided in the Supplemental Appendix (follow the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org).