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Programmed Genome Rearrangements in the Ciliate

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  • Authors: V. Talya Yerlici1, Laura F. Landweber2
  • Editors: Martin Gellert3, Nancy Craig4
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Department of Molecular Biology, Princeton University, Princeton, NJ 08544; 2: Department of Ecology and Evolutionary Biology, Princeton University, Princeton, NJ 08544; 3: National Institutes of Health, Bethesda, MD; 4: Johns Hopkins University, Baltimore, MD
  • Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
  • Received 09 May 2014 Accepted 16 May 2014 Published 05 December 2014
  • Laura Landweber, lfl@princeton.edu
image of Programmed Genome Rearrangements in the Ciliate <span class="jp-italic">Oxytricha</span>
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  • Abstract:

    The ciliate is a microbial eukaryote with two genomes, one of which experiences extensive genome remodeling during development. Each round of conjugation initiates a cascade of events that construct a transcriptionally active somatic genome from a scrambled germline genome, with considerable help from both long and small noncoding RNAs. This process of genome remodeling entails massive DNA deletion and reshuffling of remaining DNA segments to form functional genes from their interrupted and scrambled germline precursors. The use of as a model system provides an opportunity to study an exaggerated form of programmed genome rearrangement. Furthermore, studying the mechanisms that maintain nuclear dimorphism and mediate genome rearrangement has demonstrated a surprising plasticity and diversity of noncoding RNA pathways, with new roles that go beyond conventional gene silencing. Another aspect of ciliate genetics is their unorthodox patterns of RNA-mediated, epigenetic inheritance that rival Mendelian inheritance. This review takes the reader through the key experiments in a model eukaryote that led to fundamental discoveries in RNA biology and pushes the biological limits of DNA processing.

  • Citation: Yerlici V, Landweber L. 2014. Programmed Genome Rearrangements in the Ciliate . Microbiol Spectrum 2(6):MDNA3-0025-2014. doi:10.1128/microbiolspec.MDNA3-0025-2014.

Key Concept Ranking

Gene Expression and Regulation
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DNA
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Small Interfering RNA
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/content/journal/microbiolspec/10.1128/microbiolspec.MDNA3-0025-2014
2014-12-05
2017-04-25

Abstract:

The ciliate is a microbial eukaryote with two genomes, one of which experiences extensive genome remodeling during development. Each round of conjugation initiates a cascade of events that construct a transcriptionally active somatic genome from a scrambled germline genome, with considerable help from both long and small noncoding RNAs. This process of genome remodeling entails massive DNA deletion and reshuffling of remaining DNA segments to form functional genes from their interrupted and scrambled germline precursors. The use of as a model system provides an opportunity to study an exaggerated form of programmed genome rearrangement. Furthermore, studying the mechanisms that maintain nuclear dimorphism and mediate genome rearrangement has demonstrated a surprising plasticity and diversity of noncoding RNA pathways, with new roles that go beyond conventional gene silencing. Another aspect of ciliate genetics is their unorthodox patterns of RNA-mediated, epigenetic inheritance that rival Mendelian inheritance. This review takes the reader through the key experiments in a model eukaryote that led to fundamental discoveries in RNA biology and pushes the biological limits of DNA processing.

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FIGURE 1

vegetative and sexual life cycles. cells contain one somatic macronucleus and two identical, germline, micronuclei. For simplicity only one MIC is shown. divides asexually through binary fission producing clonal offspring. (1) Under starvation conditions, two cells of different mating type form pairs. (2) The diploid MIC undergoes meiosis and produces four haploid gametes. (3) Three of the haploid gametes degrade while the one remaining divides mitotically. (4) The pair exchange a copy each of these haploid micronuclei. (5) The haploid micronuclei fuse, producing a new, diploid, zygotic nucleus shown with cross hatching. This produces exconjugants with identical zygotic genomes. (6) The zygotic nucleus divides mitotically, producing two identical zygotic genomes. (7) One copy of the zygotic genome differentiates into a new soma, while the old maternal soma degrades. The developing MAC at this stage is called the “anlage” (plural anlagen). The other zygotic nucleus will maintain the new germline. doi:10.1128/microbiolspec.MDNA3-0025-2014.f1

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 2

The inferred evolutionary relationship of to other well-studied ciliate lineages and representative eukaryotic genera. Figure modified from Bracht et al. ( 77 ). Branch order, branch lengths and scale bar are based on estimates in Parfrey et al. ( 83 ) and Chang et al. ( 141 ). doi:10.1128/microbiolspec.MDNA3-0025-2014.f2

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 3

Schematic illustration of gene unscrambling in stichotrichous ciliates. Black bars represent IESs to be eliminated. These IESs separate the MDSs (shown as white arrows) in the micronuclear precursor region. MDS labeled M1-9 and drawn with dotted lines are the precursors for one somatic nanochromosome, whereas MDSs drawn with solid lines (m1-7) assemble into a separate nanochromosome. The numbers represent the order in which the MDSs appear in the mature MAC nanochromosomes. Mature nanochromosomes in the MAC form following IES excision, MDSs descrambling and chromosome fragmentation. Addition of telomeres (black tall rectangles) at the boundaries of the first and last MDS produces mature nanochromosomes. doi:10.1128/microbiolspec.MDNA3-0025-2014.f3

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 4

piRNAs mark MDSs for retention during genome rearrangement. A. 27-nucleotide piRNAs (short black bars) derive from RNA copies of the maternal somatic genome. These piRNAs mark and protect MDSs for retention. DNA segments to be eliminated, such as IESs (shown as white rectangles between MDSs in the precursor DNA segment), lack corresponding piRNAs. Direct repeats (pointers) flanking the IESs are shown as small, patterned vertical rectangles. Identical pointers that join two consecutive MDSs are marked with the same pattern. The excision machinery (represented as scissors) can cleave the regions not protected by piRNAs and permit recombination between pointers. This leads to IES elimination, with retention of one copy of the pointer between MDSs in the mature nanochromosome. Dots at the ends of precursor loci in the micronucleus and developing macronucleus represent sequences that continue beyond the regions shown. Mature, macronuclear nanochromosomes terminate in telomeres (thin, vertical, black rectangles). B. Microinjection into conjugating cells of synthetic 27-nucleotide piRNAs (short white bars) complementary to IESs that are normally deleted in wild type cells, leads to retention of the IES in the mature MAC of the exconjugants and subsequent sexual offspring ( 32 ). This creates IES strains. doi:10.1128/microbiolspec.MDNA3-0025-2014.f4

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 5a

Genome differentiation in is mediated by long noncoding maternal RNA templates. A. Nowacki et al. ( 85 ) demonstrated the transient presence of two-telomere-containing, bidirectional RNA transcripts of maternal nanochromosomes in conjugating cells. Panel A shows part of the TEBPα (Telomere End Binding Protein ) locus between segments 5 and 17 and the corresponding region of the RNA template (long thin lines). B. These templates may guide rearrangement in the anlagen by interacting with the MIC precursor DNA. Numbered black rectangles represent MDSs in the linear MAC order. White rectangles indicate nonscrambled IESs (interrupting consecutive MDSs in the MAC) and striped rectangles are scrambled IESs that map between nonconsecutive MDSs. C. RNAi targeting the maternal RNA templates (wavy line) leads to gross rearrangement defects in the corresponding gene ( 85 ). A PCR assay of the region between MDS 5 to 17 revealed the presence of molecules that are longer than wild type MAC sequences because they often retain unspliced IESs, with a strong bias towards retaining IESs between scrambled segments (examples i, ii, iv). Consistent with studies of earlier timepoints in the rearrangement cascade ( 49 ), example (ii) only eliminated a subset of the nonscrambled IESs in this region, and examples (i), (iv), (v) and (vi) eliminated nonscrambled IESs. In addition, cases (i) and (ii) stalled before any reordering. The presumed decrease in template abundance also leads to accumulation of partially unscrambled molecules, such as (iii), (iv) and (v). Case (iv) correctly repaired MDS 12 between MDS 11 and 13, but with an unexpected duplication of MDS 12, retaining a copy in its scrambled location as well. The duplications in (iv) and (vi) indicate that intermolecular DNA rearrangement may be tolerated. Aberrantly spliced junctions, marked by thin vertical ovals, occur at cryptic repeats, instead of endogenous pointers. D. Microinjection of synthetic templates with the order of segments 7 and 8 transposed (indicated with curly arrows) produces new, scrambled MAC nanochromosomes with the reprogrammed order. Wild type, unscrambled nanochromosomes coexist in the cell with the permuted chromosomes, because the endogenous wild type templates were not destroyed in this experiment ( 85 ). doi:10.1128/microbiolspec.MDNA3-0025-2014.f5

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 5b

Genome differentiation in is mediated by long noncoding maternal RNA templates. A. Nowacki et al. ( 85 ) demonstrated the transient presence of two-telomere-containing, bidirectional RNA transcripts of maternal nanochromosomes in conjugating cells. Panel A shows part of the TEBPα (Telomere End Binding Protein ) locus between segments 5 and 17 and the corresponding region of the RNA template (long thin lines). B. These templates may guide rearrangement in the anlagen by interacting with the MIC precursor DNA. Numbered black rectangles represent MDSs in the linear MAC order. White rectangles indicate nonscrambled IESs (interrupting consecutive MDSs in the MAC) and striped rectangles are scrambled IESs that map between nonconsecutive MDSs. C. RNAi targeting the maternal RNA templates (wavy line) leads to gross rearrangement defects in the corresponding gene ( 85 ). A PCR assay of the region between MDS 5 to 17 revealed the presence of molecules that are longer than wild type MAC sequences because they often retain unspliced IESs, with a strong bias towards retaining IESs between scrambled segments (examples i, ii, iv). Consistent with studies of earlier timepoints in the rearrangement cascade ( 49 ), example (ii) only eliminated a subset of the nonscrambled IESs in this region, and examples (i), (iv), (v) and (vi) eliminated nonscrambled IESs. In addition, cases (i) and (ii) stalled before any reordering. The presumed decrease in template abundance also leads to accumulation of partially unscrambled molecules, such as (iii), (iv) and (v). Case (iv) correctly repaired MDS 12 between MDS 11 and 13, but with an unexpected duplication of MDS 12, retaining a copy in its scrambled location as well. The duplications in (iv) and (vi) indicate that intermolecular DNA rearrangement may be tolerated. Aberrantly spliced junctions, marked by thin vertical ovals, occur at cryptic repeats, instead of endogenous pointers. D. Microinjection of synthetic templates with the order of segments 7 and 8 transposed (indicated with curly arrows) produces new, scrambled MAC nanochromosomes with the reprogrammed order. Wild type, unscrambled nanochromosomes coexist in the cell with the permuted chromosomes, because the endogenous wild type templates were not destroyed in this experiment ( 85 ). doi:10.1128/microbiolspec.MDNA3-0025-2014.f5

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 5c

Genome differentiation in is mediated by long noncoding maternal RNA templates. A. Nowacki et al. ( 85 ) demonstrated the transient presence of two-telomere-containing, bidirectional RNA transcripts of maternal nanochromosomes in conjugating cells. Panel A shows part of the TEBPα (Telomere End Binding Protein ) locus between segments 5 and 17 and the corresponding region of the RNA template (long thin lines). B. These templates may guide rearrangement in the anlagen by interacting with the MIC precursor DNA. Numbered black rectangles represent MDSs in the linear MAC order. White rectangles indicate nonscrambled IESs (interrupting consecutive MDSs in the MAC) and striped rectangles are scrambled IESs that map between nonconsecutive MDSs. C. RNAi targeting the maternal RNA templates (wavy line) leads to gross rearrangement defects in the corresponding gene ( 85 ). A PCR assay of the region between MDS 5 to 17 revealed the presence of molecules that are longer than wild type MAC sequences because they often retain unspliced IESs, with a strong bias towards retaining IESs between scrambled segments (examples i, ii, iv). Consistent with studies of earlier timepoints in the rearrangement cascade ( 49 ), example (ii) only eliminated a subset of the nonscrambled IESs in this region, and examples (i), (iv), (v) and (vi) eliminated nonscrambled IESs. In addition, cases (i) and (ii) stalled before any reordering. The presumed decrease in template abundance also leads to accumulation of partially unscrambled molecules, such as (iii), (iv) and (v). Case (iv) correctly repaired MDS 12 between MDS 11 and 13, but with an unexpected duplication of MDS 12, retaining a copy in its scrambled location as well. The duplications in (iv) and (vi) indicate that intermolecular DNA rearrangement may be tolerated. Aberrantly spliced junctions, marked by thin vertical ovals, occur at cryptic repeats, instead of endogenous pointers. D. Microinjection of synthetic templates with the order of segments 7 and 8 transposed (indicated with curly arrows) produces new, scrambled MAC nanochromosomes with the reprogrammed order. Wild type, unscrambled nanochromosomes coexist in the cell with the permuted chromosomes, because the endogenous wild type templates were not destroyed in this experiment ( 85 ). doi:10.1128/microbiolspec.MDNA3-0025-2014.f5

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 5d

Genome differentiation in is mediated by long noncoding maternal RNA templates. A. Nowacki et al. ( 85 ) demonstrated the transient presence of two-telomere-containing, bidirectional RNA transcripts of maternal nanochromosomes in conjugating cells. Panel A shows part of the TEBPα (Telomere End Binding Protein ) locus between segments 5 and 17 and the corresponding region of the RNA template (long thin lines). B. These templates may guide rearrangement in the anlagen by interacting with the MIC precursor DNA. Numbered black rectangles represent MDSs in the linear MAC order. White rectangles indicate nonscrambled IESs (interrupting consecutive MDSs in the MAC) and striped rectangles are scrambled IESs that map between nonconsecutive MDSs. C. RNAi targeting the maternal RNA templates (wavy line) leads to gross rearrangement defects in the corresponding gene ( 85 ). A PCR assay of the region between MDS 5 to 17 revealed the presence of molecules that are longer than wild type MAC sequences because they often retain unspliced IESs, with a strong bias towards retaining IESs between scrambled segments (examples i, ii, iv). Consistent with studies of earlier timepoints in the rearrangement cascade ( 49 ), example (ii) only eliminated a subset of the nonscrambled IESs in this region, and examples (i), (iv), (v) and (vi) eliminated nonscrambled IESs. In addition, cases (i) and (ii) stalled before any reordering. The presumed decrease in template abundance also leads to accumulation of partially unscrambled molecules, such as (iii), (iv) and (v). Case (iv) correctly repaired MDS 12 between MDS 11 and 13, but with an unexpected duplication of MDS 12, retaining a copy in its scrambled location as well. The duplications in (iv) and (vi) indicate that intermolecular DNA rearrangement may be tolerated. Aberrantly spliced junctions, marked by thin vertical ovals, occur at cryptic repeats, instead of endogenous pointers. D. Microinjection of synthetic templates with the order of segments 7 and 8 transposed (indicated with curly arrows) produces new, scrambled MAC nanochromosomes with the reprogrammed order. Wild type, unscrambled nanochromosomes coexist in the cell with the permuted chromosomes, because the endogenous wild type templates were not destroyed in this experiment ( 85 ). doi:10.1128/microbiolspec.MDNA3-0025-2014.f5

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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FIGURE 6

A model for noncoding RNA-mediated genome rearrangement in . Figure modified from Bracht et al. ( 77 ). The bidirectional transcription of the maternal MAC genome produces whole-chromosome transcripts which includes telomeres (long thin line flanked by black vertical rectangles). Either this maternal RNA template or other MAC transcripts are processed into 27-nucleotide piRNAs (short line segments) that interact with the Piwi protein, Otiwi1 (drawn as white ovals). Otiwi1 transports the piRNAs to the anlagen, in conjunction with the template RNAs. Here, piRNAs interact with the MIC precursor DNA to differentiate MDSs (black boxes) from IESs (white intervening rectangles). The template RNA then guides MDS descrambling, which, together with chromosome fragmentation and telomere addition, forms mature MAC nanochromosomes. doi:10.1128/microbiolspec.MDNA3-0025-2014.f6

Source: microbiolspec December 2014 vol. 2 no. 6 doi:10.1128/microbiolspec.MDNA3-0025-2014
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