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Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium

ABSTRACT: Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells given its unique feature of nuclear dimorphism. Here, formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.

SUBMITTER: Dr. Aditi Singh

PROVIDER: S-SCDT-EMBOJ-2022-111839 | biostudies-other |

REPOSITORIES: biostudies-other

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Chromatin remodeling is required for sRNA-guided DNA elimination in Paramecium.

Project description:Small RNAs mediate the silencing of transposable elements and other genomic loci, increasing nucleosome density and preventing undesirable gene expression. The unicellular ciliate Paramecium is a model to study dynamic genome organization in eukaryotic cells, given its unique feature of nuclear dimorphism. Here, the formation of the somatic macronucleus during sexual reproduction requires eliminating thousands of transposon remnants (IESs) and transposable elements scattered throughout the germline micronuclear genome. The elimination process is guided by Piwi-associated small RNAs and leads to precise cleavage at IES boundaries. Here we show that IES recognition and precise excision are facilitated by recruiting ISWI1, a Paramecium homolog of the chromatin remodeler ISWI. ISWI1 knockdown substantially inhibits DNA elimination, quantitatively similar to development-specific sRNA gene knockdowns but with much greater aberrant IES excision at alternative boundaries. We also identify key development-specific sRNA biogenesis and transport proteins, Ptiwi01 and Ptiwi09, as ISWI1 cofactors in our co-immunoprecipitation studies. Nucleosome profiling indicates that increased nucleosome density correlates with the requirement for ISWI1 and other proteins necessary for IES excision. We propose that chromatin remodeling together with small RNAs is essential for efficient and precise DNA elimination in Paramecium.

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The SUMO pathway is developmentally regulated and required for programmed DNA elimination in Paramecium tetraurelia.

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Structural maintenance of chromosomes (SMCs) proteins are required for developmental DNA elimination in Paramecium tetraurelia

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Chromatin remodeling is required for gene reactivation after decitabine-mediated DNA hypomethylation.

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Project description:Supply of exogenous dsRNA (exo-dsRNA), either by injection or by feeding, is a fast and powerful alternative to classical knockout studies. The biotechnical potential of feeding techniques is evident from the numerous studies focusing on oral administration of dsRNA to control pests and viral infection in crops/animal farming. We aimed to dissect the direct and indirect effects of exo-dsRNA feeding on the endogenous short interfering RNA (endo-siRNA) populations of the free-living ciliate Paramecium. We introduced dsRNA fragments against Dicer1 (DCR1), involved in RNA interference (RNAi) against exo- and few endo-siRNAs, and an RNAi unrelated gene, ND169. Any feeding, even the control dsRNA, diminishes genome wide the accumulation of endo-siRNAs and mRNAs. This cannot be explained by direct off-target effects and suggests mechanistic overlaps of the exo- and endo-RNAi mechanisms. Nevertheless, we observe a stronger down-regulation of mRNAs in DCR1 feeding compared with ND169 knockdown. This is likely due to the direct involvement of DCR1 in endo-siRNA accumulation. We further observed a cis-regulatory effect on mRNAs that overlap with phased endo-siRNAs. This interference of exo-dsRNA with endo-siRNAs warrants further investigations into secondary effects in target species/consumers, risk assessment of dsRNA feeding applications, and environmental pollution with dsRNA.

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Cis-acting signals modulate the efficiency of programmed DNA elimination in Paramecium tetraurelia.

Project description:In Paramecium, the regeneration of a functional somatic genome at each sexual event relies on the elimination of thousands of germline DNA sequences, known as Internal Eliminated Sequences (IESs), from the zygotic nuclear DNA. Here, we provide evidence that IESs' length and sub-terminal bases jointly modulate IES excision by affecting DNA conformation in P. tetraurelia. Our study reveals an excess of complementary base pairing between IESs' sub-terminal and contiguous sites, suggesting that IESs may form DNA loops prior to cleavage. The degree of complementary base pairing between IESs' sub-terminal sites (termed Cin-score) is positively associated with IES length and is shaped by natural selection. Moreover, it escalates abruptly when IES length exceeds 45 nucleotides (nt), indicating that only sufficiently large IESs may form loops. Finally, we find that IESs smaller than 46 nt are favored targets of the cellular surveillance systems, presumably because of their relatively inefficient excision. Our findings extend the repertoire of cis-acting determinants for IES recognition/excision and provide unprecedented insights into the distinct selective pressures that operate on IESs and somatic DNA regions. This information potentially moves current models of IES evolution and of mechanisms of IES recognition/excision forward.

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