Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium
- CEA, CNRS, Univ. Paris-Sud, Université Paris-Saclay, France
- Institute of Cell Biology, University of Bern, Switzerland
- Institut de Biologie de l’Ecole Normale Supérieure, France
- Univ Paris Diderot, France
Figures
Figure 1 with 2 supplements
Novel domesticated PiggyBac transposases in Paramecium.
(A) Domain organization of the PiggyBac transposase (PB) from T. ni and of Paramecium PiggyBac-related proteins (Pgm and PgmLs). The Pfam domain DDE_Tnp_1_7 is shown as a bipartite orange domain, …
Figure 1—figure supplement 1
MUSCLE alignment of the cysteine-rich domains of ciliate domesticated PB transposases and other PB transposases.
The analysis involved 62 amino acid sequences of PB transposases and domesticated transposases from ciliates and other species. Amino acid sequences encompassing the cysteine-rich domain of each …
Figure 1—figure supplement 2
Maximum Likelihood tree of ciliate domesticated PB transposases and other PB transposases.
The tree includes 69 amino acid sequences of PB transposases and domesticated PB transposases from ciliates and other species. To construct the tree, the alignment of all transposase core domains (Su…
Figure 2 with 2 supplements
Expression and nuclear localization of PgmLs during autogamy.
(A) Normalized RNA-seq data were extracted from (Arnaiz et al., 2017) and used to calculate mean expression levels for each time-point. V: vegetative cells (V1.2); S: starved cells with meiotic …
Figure 2—figure supplement 1
Validation of the specificity of antibodies directed against Pgm, PgmL1, PgmL5a, and the Flag peptide by immunofluorescence labelling of fixed cells.
(A) Immunostaining of Pgm in early autogamous cells subjected to control (ND7) or PGM RNAi. (B) Immunostaining of PgmL1 in early autogamous cells subjected to control (L4440) or PGML1 RNAi. (C) …
Figure 2—figure supplement 2
Localization of GFP and RFP fusions in developing new MACs.
Plasmids expressing GFP-PgmL1, GFP-PgmL2 and GFP-PgmL5b are derivatives of pUC19 and carry the EGFP coding sequence (Singh et al., 2014) fused to the 5’ end of each PGML coding sequence. They were …
Figure 3 with 2 supplements
PgmLs are essential during autogamy and interact with Pgm in cell extracts.
(A) Effect of PGML KDs on the recovery of post-autogamous progeny with functional new MACs. For PGML1, PGML2 and PGML3c, only the results obtained using IF1 RNAi constructs (Figure 3—figure …
Figure 3—figure supplement 1
Map and coordinates of PGML feeding inserts.
Two feeding inserts (IF1 and IF2) were designed for each gene. Within multigenic PGML groups, gene-specific inserts are in red and inserts that are able to cross-silence other genes of the same …
Figure 3—figure supplement 2
Co-precipitation of MBP-Pgm with HA-PgmL fusions.
(A) Control DRaCALA DNA binding assay (Differential Radial Capillary Action of Ligand Assay, see [Donaldson et al., 2012]). MBP-Pgm was purified from insect cells. Purified MBP-Pgm (400 nM final …
Figure 4 with 2 supplements
Expression and localization of Pgm in PGML KDs.
(A) Immunostaining of Pgm in early autogamous cells subjected to control (L4440) or PGML RNAi. Developing MACs are indicated by white arrowheads. Scale bar is 10 μm. (B) Western blot analysis of Pgm …
Figure 4—figure supplement 1
Plot of Pgm mean immunofluorescence intensity vs developing MAC size in cells subjected to control or PGML RNAi.
For each RNAi condition, 44 to 48 developing MACs were analyzed on slides carrying whole cells immunostained with α-Pgm antibodies (Figure 4). The Pgm mean fluorescence intensity was plotted against …
Figure 4—figure supplement 2
Immunolocalization of Pgm without Triton extraction in PGML knockdowns.
(A) Immunostaining of Pgm in cells subjected to control (L4440), PGML or PGM RNAi. Cells were fixed for 10 min in PHEM +2% formaldehyde, permeabilized for 15 min in PHEM +1% Triton before TBST +3% …
Figure 5 with 3 supplements
Analysis of IES retention in PGML KDs.
(A) Distribution of IES retention scores (IRS) in PGML KDs. Grey bars represent the distribution of all IESs over IRS ranging from 0 to 1 (by bins of 0.025). The distribution obtained in a …
Figure 5—figure supplement 1
Northern blot analysis of PGML mRNA during autogamy in PGML knockdowns.
Top panels: Detection of PGML mRNAs by northern blot hybridization. RNAi against the ND7 gene was used as a control. PGML1 and PGML2 genes were knocked down individually using their respective IF1 …
Figure 5—figure supplement 2
Analysis of IES retention scores in partial PGML2 KDs.
(A) Distribution of IES retention scores in complete and partial PGML2 KDs. Dilutions of PGML2 RNAi-inducing bacteria are indicated in each panel. For each condition, the distribution of …
Figure 5—figure supplement 3
Correlation between IES retention scores in PGM, PGML and partial PGML2 knockdowns.
The distributions of IES boundary scores are displayed in the diagonal for each knockdown. For each pair of KDs, level plots of IES retention scores were drawn using the ‘ggplot2’ package (Wickham, …
Figure 6 with 4 supplements
IES excision errors in PGML KDs.
(A) Number of IES excision errors in the MAC of vegetative cells before autogamy (No KD) and in the new MAC of autogamous cells upon each PGML KD (de novo errors). For the No KD sample, the error …
Figure 6—figure supplement 1
The number of excision errors increases for IESs with the lowest retention scores in PGML1 or PGML3a and b knockdowns.
(A) Fraction of IESs with errors as a function of boundary scores. For each dataset (see Supplementary file 9), the distribution of IES boundary scores is shown in grey (interval width: 0.025). For …
Figure 6—figure supplement 2
Raw counts of IES excision errors in PGML1, PGML3a and b and partial PGML2 knockdowns.
(A) Raw counts of IES excision errors in PGML1, PGML3a and b and partial PGML2 KDs. Here, and in contrast to Figure 6C, the distribution of different classes of IES excision errors for each …
Figure 6—figure supplement 3
Alternative excision boundaries used in partial internal IES excision errors.
(A) Fraction of IES ends with a TA dinucleotide localized at each indicated distance from the reference TA boundary. Position 0 corresponds to the T of the reference TA at each IES end. (B) SeqLogos …
Figure 6—figure supplement 4
Size distribution of IESs with partial internal excision errors in PGML1 or PGML3a and b KDs.
(A) Size distribution of IESs exhibiting partial internal errors in a PGML1 KD. (B) Same as panel A for a PGML3a and b KD. (C) For each IES size, the black curve shows the fraction of IESs relative …
Figure 7
Model for IES excision mediated by a multicomponent Pgm/PgmLs complex.
This figure summarizes the observed effects of PGML KDs on Pgm-mediated IES excision. In line with previously published data (Dubois et al., 2017) and known properties of the T. ni PiggyBac …
Author response image 1
Paramecium proteins are highly divergent relative to their mammalian orthologs.
Percent identity between orthologs was calculated using the InParanoid tool from Cildb (Arnaiz et al., 2009, Database 2000 bap022; Arnaiz et al., 2014, Cilia 3:9; http://cildb.cgm.cnrs-gif.fr/). …
Author response image 2
Attempts to extract endogenous Pgm from chromatin in control cells.
Paramecium nuclei were purified by low-speed centrifugation of autogamous cell lysates, as described (Arnaiz et al., 2012). Immunoblotting was performed using α-Pgm 2659-GP antibodies for Pgm …
Additional files
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Supplementary file 1
MUSCLE alignment of the transposase core domains of ciliate domesticated PB transposases and other PB transposases
- https://doi.org/10.7554/eLife.37927.024
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Supplementary file 2
Table of Pgm and PgmL proteins encoded by published Paramecium genomes and their ParameciumDB accession numbers
- https://doi.org/10.7554/eLife.37927.025
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Supplementary file 3
Sequences of the cysteine-rich domains used for the alignment shown in Figure 1—figure supplement 1
- https://doi.org/10.7554/eLife.37927.026
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Supplementary file 4
Sequences of the transposase core domains used for the alignement shown in Supplementary file 1.
- https://doi.org/10.7554/eLife.37927.027
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Supplementary file 5
Sequence of the synthetic PGML genes used for protein production in insect cells
- https://doi.org/10.7554/eLife.37927.028
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Supplementary file 6
Analysis of post-autogamous progeny in small-scale PGML knockdowns,
- https://doi.org/10.7554/eLife.37927.029
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Supplementary file 7
Analysis of post-autogamous progeny in middle- and large-scale PGML knockdowns
- https://doi.org/10.7554/eLife.37927.030
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Supplementary file 8
DNA-seq datasets from ENA project PRJEB24171 (this study)
- https://doi.org/10.7554/eLife.37927.031
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Supplementary file 9
Analysis of IES excision reads in PGM and PGML knockdowns
- https://doi.org/10.7554/eLife.37927.032
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Transparent reporting form
- https://doi.org/10.7554/eLife.37927.033
