scholarly journals Characterization of the RNA-interference pathway as a tool for reverse genetic analysis in the nascent phototrophic endosymbiosis, Paramecium bursaria

2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Benjamin H. Jenkins ◽  
Finlay Maguire ◽  
Guy Leonard ◽  
Joshua D. Eaton ◽  
Steven West ◽  
...  
Keyword(s):  
Rna Interference ◽  
Paramecium Bursaria ◽  
Rnai Pathway ◽  
Reverse Genetic ◽  
Knock Down ◽  
Protein Encoding ◽  
Host Growth ◽  
Rna Pathways ◽  
Si Rna

Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote–eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1 , which we show to be crucial to host growth. Finally, using simultaneous knock-down ‘paradox’ controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1 , Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria – Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.

scholarly journals Characterisation of the RNA-interference pathway as a Tool for Genetics in the Nascent Phototrophic Endosymbiosis, Paramecium bursaria

2020 ◽  
Author(s):  
Benjamin H. Jenkins ◽  
Finlay Maguire ◽  
Guy Leonard ◽  
Joshua D. Eaton ◽  
Steven West ◽  
...  
Keyword(s):  
Rna Interference ◽  
Genetic Basis ◽  
Paramecium Bursaria ◽  
Rnai Pathway ◽  
Knock Down ◽  
Protein Encoding ◽  
Host Growth ◽  
Rna Pathways ◽  
Si Rna ◽  
Encoding Genes

ABSTRACTEndosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote-eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria–Chlorella spp. Using comparative genomics and transcriptomics supported by phylogentics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA, and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We then validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1, which we show to be crucial to host growth. Finally, using simultaneous knock-down paradox controls to rescue the effect u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1, Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating use of the P. bursaria-Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.

scholarly journals Selection and Characterization of RNA Interference-Deficient Trypanosomes Impaired in Target mRNA Degradation

2004 ◽  
Vol 3 (6) ◽  
pp. 1445-1453 ◽  
Author(s):  
Huafang Shi ◽  
Nathalie Chamond ◽  
Christian Tschudi ◽  
Elisabetta Ullu
Keyword(s):  
Rna Interference ◽  
Biological Significance ◽  
Cellular Level ◽  
Rnai Pathway ◽  
Small Interfering Rnas ◽  
Wild Type ◽  
Double Stranded Rna ◽  
Target Mrna ◽  
Insight Into

ABSTRACT Genetic analysis of the RNA interference (RNAi) pathway in Trypanosoma brucei has so far revealed one essential component, namely, TbAGO1, encoding a member of the Argonaute protein family. To gain further insight into the RNAi mechanism and its biological significance, we selected RNAi-deficient trypanosomes by using repeated cycles of electroporation with α-tubulin double-stranded RNA, a treatment that blocks cytokinesis in wild-type cells. Two independent clones, termed RiD-1 (for RNAi-deficient clone 1) and RiD-2, were characterized. At the cellular level, only RiD-1 trypanosomes showed a significant increase in doubling time with the concomitant accumulation of cells defective in the completion of cytokinesis. At the RNA level, both clones accumulated wild-type amounts of small interfering RNAs and displayed elevated levels of retroposon transcripts, the hallmark of RNAi deficiency in T. brucei. Importantly, both RiD-1 and RiD-2 clones were defective in the degradation of target mRNA, suggesting an impairment of the activity of AGO1, the putative RNAi endonuclease. Since in RiD cells the AGO1 gene was not mutated and was expressed at wild-type levels, we propose that in trypanosomes the cleavage of mRNA by AGO1 is regulated by the interaction with another factor(s).

scholarly journals Construction and characterization of a knock-down RNA interference line of OsNRPD1 in rice ( Oryza sativa ssp japonica cv Nipponbare)

2020 ◽  
Vol 375 (1795) ◽  
pp. 20190338 ◽  
Author(s):  
Emilie Debladis ◽  
Tzuu-Fen Lee ◽  
Yan-Jiun Huang ◽  
Jui-Hsien Lu ◽  
Sandra M. Mathioni ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Oryza Sativa ◽  
Rna Interference ◽  
Rice Genome ◽  
Small Interfering Rnas ◽  
In Planta ◽  
Knock Out ◽  
Knock Down ◽  
Pol Iv

In plants, RNA-directed DNA methylation (RdDM) is a silencing mechanism relying on the production of 24-nt small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) to trigger methylation and inactivation of transposable elements (TEs). We present the construction and characterization of osnrpd1 , a knock-down RNA interference line of OsNRPD1 gene that encodes the largest subunit of Pol IV in rice ( Oryza sativa ssp japonica cv Nipponbare). We show that osnrpd1 displays a lower accumulation of OsNRPD1 transcripts, associated with an overall reduction of 24-nt siRNAs and DNA methylation level in all three contexts, CG, CHG and CHH. We uncovered new insertions of known active TEs, the LTR retrotransposons Tos17 and Lullaby and the long interspersed nuclear element-type retrotransposon Karma. However, we did not observe any clear developmental phenotype, contrary to what was expected for a mutant severely affected in RdDM. In addition, despite the presence of many putatively functional TEs in the rice genome, we found no evidence of in planta global reactivation of transposition. This knock-down of OsNRPD1 likely led to a weakly affected line, with no effect on development and a limited effect on transposition. We discuss the possibility that a knock-out mutation of OsNRPD1 would cause sterility in rice. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

scholarly journals Emergent RNA-RNA interactions can promote stability in a nascent phototrophic endosymbiosis

2021 ◽  
Author(s):  
Benjamin H Jenkins ◽  
Finlay Maguire ◽  
Guy Leonard ◽  
Joshua D Eaton ◽  
Steven West ◽  
...  
Keyword(s):  
Rna Processing ◽  
Rnai Pathway ◽  
Knock Down ◽  
Sequence Identity ◽  
Host Growth ◽  
Multiple Regions ◽  
Enforcement Mechanisms ◽  
Genetic Integration ◽  
Host Genes ◽  
Biological Organisms

Eukaryote-eukaryote endosymbiosis was responsible for the spread of photosynthetic organelles. Interaction stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms which act to stabilise nascent endosymbioses - between two fundamentally selfish biological organisms - are unclear. Theory suggests that enforcement mechanisms, which punish misbehaviour, may act to stabilise such interactions by resolving conflict. However, how such mechanisms can emerge in a nascent eukaryote-eukaryote endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of endosymbionts, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model nascent endosymbiosis, Paramecium bursaria - Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) pathway. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived mRNA released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We additionally identify multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate, through delivery of synthetic endosymbiont sRNA, that exposure to these regions can knock-down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knock-down in response to endosymbiont-derived RNA processing by the host, which we term 'RNAi-collisions', represents a mechanism which can promote stability in a nascent eukaryote-eukaryote endosymbiosis. By imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of a symbiosis across fluctuating ecologies and symbiotic states.

scholarly journals Isolation and Characterization of Strain Exiguobacterium sp. KRL4, a Producer of Bioactive Secondary Metabolites from a Tibetan Glacier

2021 ◽  
Vol 9 (5) ◽  
pp. 890
Author(s):  
Pietro Tedesco ◽  
Fortunato Palma Esposito ◽  
Antonio Masino ◽  
Giovanni Andrea Vitale ◽  
Emiliana Tortorella ◽  
...  
Keyword(s):  
Phenotypic Analysis ◽  
Biological Assays ◽  
C Elegans ◽  
Isolation And Characterization ◽  
Wide Range ◽  
Protein Encoding ◽  
Nematocidal Activity ◽  
Extremophilic Microorganisms ◽  
Unique Source

Extremophilic microorganisms represent a unique source of novel natural products. Among them, cold adapted bacteria and particularly alpine microorganisms are still underexplored. Here, we describe the isolation and characterization of a novel Gram-positive, aerobic rod-shaped alpine bacterium (KRL4), isolated from sediments from the Karuola glacier in Tibet, China. Complete phenotypic analysis was performed revealing the great adaptability of the strain to a wide range of temperatures (5–40 °C), pHs (5.5–8.5), and salinities (0–15% w/v NaCl). Genome sequencing identified KRL4 as a member of the placeholder genus Exiguobacterium_A and annotation revealed that only half of the protein-encoding genes (1522 of 3079) could be assigned a putative function. An analysis of the secondary metabolite clusters revealed the presence of two uncharacterized phytoene synthase containing pathways and a novel siderophore pathway. Biological assays confirmed that the strain produces molecules with antioxidant and siderophore activities. Furthermore, intracellular extracts showed nematocidal activity towards C. elegans, suggesting that strain KRL4 is a source of anthelmintic compounds.

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