RNAi pathway components and function in Paramecium bursaria Finlay - - PowerPoint PPT Presentation

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RNAi pathway components and function in Paramecium bursaria Finlay Maguire University of Exeter June 2, 2016 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview

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RNAi pathway components and function in Paramecium bursaria

Finlay Maguire

University of Exeter

June 2, 2016

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Overview

Motivation RNAi in ciliates Experimental RNAi induction in P. bursaria RNAi pathway components in active P. bursaria transcriptome(s) In-silico analysis of potential endosymbiont ‘cross-talk’ Conclusions

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Why is Paramecium bursaria potentially a good model for (secondary photosynthetic) endosymbiosis?

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Broad diversity of plastid endosymbioses

Reproduced from [Arc09].

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Paramecium bursaria and its green algal endosymbionts

▶ 100 µm to 160 µm serial phagotrophic ciliate (nuclear

dimorphism).

▶ ∼ 300 endosymbiotic algae in stable heritable facultative(?)

endosymbiosis.

▶ Multiple independent origins of these endosymbioses. ▶ Single cell transcriptome and genome of P.

bursaria-Micractinium reisseri CCAP 1660/12.

▶ P. bursaria bulk transcriptome Yad1g1N [KSD+14].

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RNAi pathways in the ciliates

Tetrahymena thermophila Oxytricha trifallax Paramecium bursaria Paramecium caudatum Paramecium multimicronucleatum Paramecium sexaurelia Paramecium primaurelia Paramecium octaurelia Paramecium tredecaurelia Paramecium tetaurelia

Paramecium aurelia species complex

?

A good model needs a means to test hypotheses:

▶ Ciliate specific scnRNA system [MG04, CMM13]. ▶ siRNA pathways present in Paramecium tetaurelia

[GS01, GS02] (and Tetrahymena thermophila [CL06, YC05]):

  • 1. Transgene inducible pathway [GS01].
  • 2. Exogenous dsRNA inducible pathway (feeding or injection)

[GS02].

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Transgene pathway

▶ Microinjection and transformation of MAC with high-copy

transgenes lacking 3’ UTR [GS01]:

  • 1. 23nt siRNA generated from transgene transcripts (Dcr1, Rdr2,

Rdr3 and Cid2) [LNS+09, MCT+14].

  • 2. mRNA cleavage (Ptiwi13 and Ptiwi14).
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Exogenous dsRNA pathway

Reproduced from [CGA+15].

▶ Exogenous dsRNA via

feeding (or microinjection) [GS02].

▶ 1◦ siRNA targeted cleavage

(Ptiwi13) [BGK+11].

▶ Undefined role in MAC for

2◦ siRNA (Ptiwi12, Ptiwi15) [MCT+14, CGA+15, BGK+11].

▶ Pds1 involved in uptake of

dsRNA from vacuole? [CGA+15].

▶ Activated at low levels by

ssRNA from normal food bacteria [CGA+15].

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So, can we experimentally induce RNAi in P. bursaria?

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Experimental feeding vector

L4440 2790 bp 698 466 233 2790 2558 2325 2093 1860 1628 1396 1163 931 pBR322_origin ORF frame 2 Ampicillin AmpR_promoter f1_origin lacZ_a T7_promoter T7_promoter Insert

Transformed into E. coli with IPTG-inducible T7 polymerase and RNAse III deficiency.

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Construct inserts

Gene Function RNAi phenotype in P. tetaurelia epi2 Epiplasmin “Monstrous” cells NSF Membrane fusion factor Lethal bug22 Basal body/ciliary protein Slow swimming and death

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RNAi feeding had mixed results

500 1000 1500 2000

Cells per ml Construct = Control Construct = bug22

2 4 6 8 10

Day

500 1000 1500 2000

Cells per ml Construct = epi2

2 4 6 8 10

Day Construct = NSF

Organism

  • P. tetaurelia

CCAP 1660/12 Yad1g1N HA1 186b

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Are all the known RNAi pathway components present in the active transcriptome(s)?

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Summary of known RNAi components

Pathway Component Function transgene-induced siRNA Rdr3 RdRP Ptiwi14 Piwi both pathways Rdr2 RdRP Dcr1 Dicer Ptiwi13 Piwi Cid2 Nucleotidyl transferase exogenous dsRNA-induced siRNA Rdr1 RdRP Cid1 Nucleotidyl transferase Ptiwi12 Piwi Ptiwi15 Piwi Pds1 Import of dsRNA?

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Cid ancestor

Cid

Tetrahymena thermophila [XP̲001012854.1] Tetrahymena thermophila [XP̲001012858.1] Paramecium bursaria SW1 [comp3906̲seq0̲m.68533] Paramecium bursaria SW1 [comp3906̲seq0̲m.68531] Paramecium bursaria Yad1g [TR17851̲c0̲g1̲i8̲m.235761] Paramecium bursaria Yad1g [TR432̲c1̲g1̲i2̲m.4057]

80.7%/0.93

Paramecium biaurelia [PBIGNP33303] Paramecium tetaurelia Cid3 [GSPATP00025353001] Paramecium sexaurelia [PSEXPNG26288]

89.8%/0.94

Paramecium multimicronucleatum [PMMNP07604]

99.8%/1.00

Paramecium caudatum [PCAUDP10462]

91%/0.93

Paramecium tetaurelia Cid1 (Marker, 2014) [PTETP9100013001] Paramecium biaurelia [PBIGNP26212] Paramecium primaurelia [PPRIMP23072]

5%/0.51

Paramecium sexaurelia [PSEXPNG26738]

42%/0.71

Paramecium multimicronucleatum [PMMNP02964]

98.9%/0.99

Paramecium caudatum [PCAUDP15935]

55.4%/0.63 99.7%/1.00 59.5%/0.67 100%/1.00 97.9%/1.00

Paramecium caudatum [PSEXPNG26858] Paramecium multimicronucleatum [PMMNP03007] Paramecium sexaurelia [PSEXPNG26858] Paramecium primaurelia [PPRIMP27560] Paramecium biaurelia [PBIGNP11073] Paramecium tetaurelia Cid2 (Marker, 2014) [PTETP13400003001]

84.1%/0.91 83%/0.88 95.3%/0.96 83.9%/0.88 59.1%/0.54 99.7%/1.00 86.7%/0.69 100%/1.00

0.2 Cid2 Cid1 Cid3 Cid1-3 Ancestor? Tetrahymena thermophila Oxytricha trifallax Paramecium bursaria Paramecium caudatum Paramecium multimicronucleatum Paramecium sexaurelia Paramecium primaurelia Paramecium octaurelia Paramecium tredecaurelia Paramecium tetaurelia ?

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Pds1 absent

Pds1

0.2

Paramecium caudatum [PCAUDP0810] Paramecium sexaurelia [PSEXPNG04218] Paramecium tetaurelia Psd1 (Marker, 2014) [PTETP600032001] Paramecium primaurelia [PPRIMP00625] Paramecium multimicronucleatum [PMMNP02700] Paramecium biaurelia [PBIGNP01684] Paramecium multimicronucleatum [PMMNP02686]

100%/1.0 99.5%/0.99 31.2%/0.64 87.2%/0.93

Tetrahymena thermophila Oxytricha trifallax Paramecium bursaria Paramecium caudatum Paramecium multimicronucleatum Paramecium sexaurelia Paramecium primaurelia Paramecium octaurelia Paramecium tredecaurelia Paramecium tetaurelia ?

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Presence/absence of known pathway components

Paramecium sexaurelia Paramecium biaurelia Paramecium caudatum Paramecium multimicronucleatum Paramecium primaurelia Paramecium tetaurelia Paramecium bursaria Rdr3 Rdr2 Ptiwi14 Cid1 Rdr1 Pds1 Ptiwi12 Ptiwi15

Presence of homologue Absence of homologue Unresolved Putative unduplicated ancestral orthologue

Ptiwi13 Dcr1 Cid2 Exogenous dsRNA factors Transgene dsRNA Required by both

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Putative RNAi component evolution scenario

Duplication of Cid1-Cid3 ancestor into Cid1 and Cid3 Duplication of ancestral Rdr into Rdr1 and Rdr2 Whole genome duplication A B? C Duplication of ancestral Cid into Cid2 and Cid1-Cid3 ancestor Tetrahymena thermophila Oxytricha trifallax Paramecium bursaria Paramecium caudatum Paramecium multimicronucleatum Paramecium sexaurelia Paramecium primaurelia Paramecium tetaurelia

Paramecium aurelia species complex

? Paramecium biaurelia Dcr1 Ancestral Rdr Ancestral Cid Ancestral Piwis B? A Rdr3 Pds1 B? C

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Could having a eukaryotic endosymbiont and RNAi activated by dsRNA in vacuoles be deleterious?

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Higher level of collisions with eukaryotes

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Collisions are a function of transcriptome size

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Conclusions

▶ RNAi phenotypes not inducible in most P. bursaria strains via

feeding.

▶ P. bursaria lacks Pds1 (in active transcriptome) thus may be

unable to take up RNA from digestive vacuoles.

▶ High levels of 23-mer collisions between P. bursaria and

eukaryotic endosymbiont transcriptomes may lead to deactivation of dsRNA uptake from vacuoles.

▶ Presence of other factors in active transcriptomes of P.

bursaria indicate transgene and microinjected exogenous dsRNA pathways may function.

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Acknowledgements

▶ Ben Jenkins (feeding experiments) ▶ David Milner (labwork) ▶ Tom Richards (PI) ▶ NHM-UCL PhD Studentship (main funding)

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References

[Arc09] John M Archibald. The puzzle of plastid evolution. Curr. Biol., 19(2):R81–8, January 2009. [BGK+11]

  • K. Bouhouche, J.-F. Gout, a. Kapusta, M. Betermier, and E. Meyer. Functional

specialization of Piwi proteins in Paramecium tetraurelia from post-transcriptional gene silencing to genome remodelling. Nucleic Acids Res., 39(10):4249–4264, 2011. [CGA+15]

  • Q. Carradec, U. Gotz, O. Arnaiz, J. Pouch, M. Simon, E. Meyer, and S. Marker. Primary

and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia. Nucleic Acids Res., 43(3):1818–1833, 2015. [CL06] Kathleen Collins and Suzanne R Lee. Two classes of endogenous small RNAs in Tetrahymena thermophila. Genes Dev., 20:28–33, 2006. [CMM13] Douglas L Chalker, E. Meyer, and Kazufumi Mochizuki. Epigenetics of ciliates. Cold Spring

  • Harb. Prespectives Epigenetics, 5:a017764, 2013.

[GS01] Angélique Galvani and Linda Sperling. Transgene-mediated post-transcriptional gene silencing is inhibited by 3 non-coding sequences in Paramecium. Nucleic Acids Res., 29(21):4387–4394, 2001. [GS02] Angélique Galvani and Linda Sperling. RNA interference by feeding in Paramecium. Trends Genet., 18(1):11–2, January 2002. [KSD+14] Yuuki Kodama, Haruo Suzuki, Hideo Dohra, Manabu Sugii, Tatsuya Kitazume, Katsushi Yamaguchi, Shuji Shigenobu, and Masahiro Fujishima. Comparison of gene expression of Paramecium bursaria with and without Chlorella variabilis symbionts. BMC Genomics, 15:183, 2014. [LNS+09]

  • G. Lepere, M. Nowacki, V. Serrano, J.-F. Gout, G. Guglielmi, S. Duharcourt, and E. Meyer.

Silencing-associated and meiosis-specific small RNA pathways in Paramecium tetraurelia. Nucleic Acids Res., 37(3):903–915, 2009. [MCT+14] Simone Marker, Quentin Carradec, Véronique Tanty, Olivier Arnaiz, and Eric Meyer. A forward genetic screen reveals essential and non-essential RNAi factors in Paramecium

  • tetraurelia. Nucleic Acids Res., 42(11):7268–7280, 2014.

[MG04] Kazufumi Mochizuki and Martin a. Gorovsky. Conjugation-specific small RNAs in Tetrahymena have predicted properties of scan (scn) RNAs involved in genome

  • rearrangement. Genes Dev., 18(Nanney 1974):2068–2073, 2004.

[YC05] Meng-Chao Yao and Ju-Lan Chao. RNA-guided DNA deletion in Tetrahymena: an RNAi-based mechanism for programmed genome rearrangements. Annu. Rev. Genet., 39:537–59, 2005.

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Microinjection proved difficult

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Dcr1

Paramecium bursaria SW1 [comp4263_seq4_m.73243] Paramecium bursaria Yad1g [TR10970_c0_g1_i3_m.150314] Paramecium multimicronucleatum [PMMNP06401] Paramecium caudatum [PCAUDP17596] Paramecium sexaurelia [PSEXPNG07454] Paramecium primaurelia [PPRIMP21835] Paramecium tetaurelia Dcr1 (Marker, 2014) [GSPATP00021751001] Paramecium biaurelia [PBIGNP07629] 14.2%/0.41 100%/0.99 100%/1.00 30.8/50 100%/1.00 100%/1.00 Oxytricha trifallax [Contig17740.0.g110_protein] Tetrahymena thermophila [gi_50897085_dbj_BAD34723.1] 100%/1.00 0.5

Dcr1

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Rdr1 Rdr2

Rdr1 & Rdr2

Tetrahymena thermophila [XP_001026321.2] Oxytricha trifallax [gi403346586] Paramecium bursaria Yad1g [TR10997_c0_g2_i5_m.152701] Paramecium bursaria SW1 [comp1394_seq3_m.27654] Paramecium bursaria Yad1g [TR24504_c0_g1_i1_m.344314] Paramecium bursaria SW1 [comp2093_seq0_m.40761] Paramecium biaurelia [PBIGNP22852] Paramecium primaurelia [PPRIMP16453] Paramecium tetaurelia Rdr2 (Marker, 2014) [GSPATP00036857001]

80.4%/0.82

Paramecium sexaurelia [PSEXPNG32890]

99.4%/0.97

Paramecium sexaurelia [PSEXPNG31119]

87.6%/0.83

Paramecium multimicronucleatum [PMMNP14096]

100%/1.00

Paramecium caudatum [PCAUDP11670]

93.3%/0.94 100%/1.00 100%/1.00

Paramecium biaurelia [PBIGNP28717] Paramecium tetaurelia Rdr1 (Marker, 2014) [PTETP8500012001] Paramecium primaurelia [PPRIMP21923]

64.1%/0.81

Paramecium sexaurelia [PSEXPNG23839]

98.5%/0.98 96.7%/0.99

Paramecium caudatum [PCAUDP07869] Paramecium multimicronucleatum [PMMNP15732]

24.6%/0.5 99.9%/1.00 100%/1.00 100%/1.00 76.7%/0.85 99.2%

0.2

/0.99

Rdr1 Rdr2 Rdr1?

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Rdr3

Paramecium bursaria Yad1g [TR463_c0_g2_i1_m.5849] Paramecium bursaria SW1 [comp11757_seq0_m.132993] Paramecium biaurelia [BIGNP03905] Paramecium primaurelia [PPRIMP22734] Paramecium tetaurelia Rdr3 (Marker, 2014) [GSPATP00006401001] 17.7%/0.50 Paramecium sexaurelia [PSEXPNG06491] 98.4%/0.96 Paramecium multimicronucleatum [PMMNP00936] 100%/1.00 Paramecium caudatum [PCAUDP10550] 96.6%/0.92 100%/1.00 0.2

Rdr3

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Psd1 Structure