The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation
- University of Copenhagen, Copenhagen Plant Science Center, Denmark
- University of Copenhagen, Department of Biology, Ole Maaløes Vej 5, Denmark
- Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Austria
- Laboratory of Molecular Biology, Wageningen University, Netherlands
Figures
Figure 1 with 2 supplements
Identification of ECT3 targets using HyperTRIBE.
(A) Phenotypes of wild type, ect3-1, and te234 (ect2-1/ect3-1/ect4-2) mutants with (lower panels) or without (upper panels) ECT3pro:ECT3-FLAG-DmADARE488Qcd-ECT3ter (ECT3-FLAG-ADAR) or ECT3pro:FLAG-Dm…
Figure 1—figure supplement 1
Identification of ECT3 targets using HyperTRIBE (extended data).
(A) Expression levels of ECT3-FLAG-ADAR or FLAG-ADAR in apices (aerial tissues) or root tips of 10-day-old seedlings of the 5+5+5 independent transgenic lines (L1–L5) used for experiments in this …
Figure 1—figure supplement 2
Characteristics of ECT3-HyperTRIBE editing sites relative to target expression levels.
(A–C) Number of significant editing sites (A), maximum or average editing proportions (B), and significance of editing sites according to either minimum or average −log10(adjusted p-value) per gene …
Figure 2 with 2 supplements
ECT3 targets are fewer and largely contained within ECT2 targets.
(A, B) Left panels: overlap of ECT2-HT and ECT3-HT targets, for the set of commonly expressed genes, in aerial (A) and root (B) tissues. Right panels: Scatter plots showing the editing proportions …
Figure 2—figure supplement 1
Sequencing depth of ECT2 and ECT3 HyperTRIBE RNA-seq data.
(A, B) Number of input reads (A) and uniquely mapped reads (B) for ECT2-HT and ECT3-HT RNA-seq samples.
Figure 2—figure supplement 2
ECT2 and ECT3 target each other and themselves.
Distribution of ECT2/3-HT editing sites and ECT2-iCLIP peaks along ECT2/3/4-encoding transcripts. ECT2 is a target of ECT3, particularly clearly in roots, and ECT3 is a target of ECT2 according to …
Figure 3 with 2 supplements
Redundancy between ECT2 and ECT3.
(A) Scatterplots comparing the editing proportions of ECT2- and ECT3-FLAG-ADAR observed in triple versus single mutant backgrounds in aerial and root tissues. They include all positions …
Figure 3—figure supplement 1
Expression levels (TPM) of the FLAG-ADAR-containing transgenes in all HyperTRIBE lines.
TPMs for the five lines of every type have been averaged for simplicity. Notice that the expression of ECT2/3-FLAG-ADAR transgenes is highly comparable in triple and single mutant backgrounds, …
Figure 3—figure supplement 2
Overlap between ECT2/3-HT targets in single and triple mutant backgrounds in roots.
Venn diagrams (left) and Upset plot (right) showing the overlap between the ECT2-HT (Arribas-Hernández et al., 2021) and ECT3-HT target sets (in single mutant backgrounds) with the groups of genes …
Figure 4 with 2 supplements
ECT2 and ECT3 targets are co-expressed with ECT2 and ECT3 in proliferating cells and enriched in biosynthetic processes.
(A) Overlap between ECT2-iCLIP target genes with ECT2-HT and ECT3-HT target gene sets. Regions outlined in bold orange and red indicate the defined permissive and stringent ECT2/3 target sets in …
Figure 4—figure supplement 1
ECT2 and ECT3 target sets in aerial and root tissues (separately).
(A, B) Overlap between HyperTRIBE target sets of ECT2 and ECT3 in aerial (A) or roots (B) tissues, and ECT2-iCLIP (whole seedlings). Genes contained within the outlined sets in roots constitute the …
Figure 4—figure supplement 2
ECT2/ECT3 targets are co-expressed with ECT2/3 in highly dividing root cells.
(A) t-SNE plot for scRNA-seq data in roots from Denyer et al., 2019, with cells colored according to their cell-type cluster definitions described on the right side (https://www.zmbp-resources.uni-tu…
Figure 5 with 2 supplements
Poly(A) sites in ECT2/3 targets do not change upon loss of ECT2/3/4 function.
(A) Experimental design. The experiment was performed once, using three biological replicates (independent lines) per group (genotype). (B) Expression pattern of ECT2-mCherry in root tips of ect2-1 …
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Figure 5—source data 1
Original (uncropped) membrane from Figure 5C.
- https://cdn.elifesciences.org/articles/72377/elife-72377-fig5-data1-v2.zip
Figure 5—figure supplement 1
FACS-sorting of root protoplasts expressing ECT2-mCherry.
Figure 5—figure supplement 2
Poly(A) sites do not change in ECT2/3 targets upon loss of ECT2/3/4 function (extended data).
(A) Summary of poly(A)-containing reads (i.e., reads with at least nine untemplated As) after removal of reads mapping upstream of purine-rich sites. These reads were used for cluster …
Figure 6 with 1 supplement
ECT2 is not in the nucleus.
(A) Standard confocal microscopy of root cells co-expressing ECT2-mCherry and GFP-WIP1. White arrows indicate areas in which apparent spills of ECT2-mCherry signal into the nucleus overlap with …
Figure 6—figure supplement 1
Super-resolution confocal microscopy of cells co-expressing ECT2-mCherry and the nuclear envelope marker GFP-WIP1.
(A) Standard confocal microscopy of root cells co-expressing ECT2-mCherry and GFP-WIP1 as in Figure 6A. (B–D) Airyscan super-resolution confocal microscopy of root cells as in (A). White dashed …
Figure 7 with 1 supplement
ECT2/3 targets are generally less abundant in cells without ECT2/3/4 function.
(A) Scatterplot of TPM expression values in Smart-seq2 libraries of root protoplasts expressing ECT2-mCherry in te234/ECT2W464A-mCherry versus ect2-1/ECT2-mCherry samples. (B) Volcano plots reveal …
Figure 7—figure supplement 1
Differential expression analysis using intact root tips of ect2/ect3/ect4 knockout plants recapitulates the results obtained with sorted protoplasts.
(A) Principal component analysis of transcriptome expression values (TPM) in Smart-seq2 libraries from FACS-sorted root protoplasts of te234/ECT2W464A-mCherry and ect2-1/ECT2-mCherry 5-day-old …
Figure 8
miRNA profile in root tips of ect2/ect3/ect4 knockout plants.
(A) Differential expression analysis of te234 versus Col-0 WT in 4-day-old root tips shows that expression of several stress-related NF-YA transcription factors (miR169defg targets) is induced in te2…
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | ECT2 | TAIR10 | AT3G13460 | EVOLUTIONARILY CONSERVED C-TERMINAL REGION 2 |
| Gene (Arabidopsis thaliana) | ECT3 | TAIR10 | AT5G61020 | EVOLUTIONARILY CONSERVED C-TERMINAL REGION 3 |
| Gene (Arabidopsis thaliana) | ECT4 | TAIR10 | AT1G55500 | EVOLUTIONARILY CONSERVED C-TERMINAL REGION 4 |
| Gene (Arabidopsis thaliana) | WIP1 | TAIR10 | AT4G26455 | WPP DOMAIN INTERACTING PROTEIN 1 |
| Gene (Drosophila melanogaster) | ADAR Isoform N | Genebank, FlyBase, NCBI | CG12598 NM_001297862 | Adenosine deaminase acting on RNA |
| Genetic reagent (A. thaliana) | SALK_002225 C (ect2-1) | NASC | N657472, N2110120 | |
| Genetic reagent (A. thaliana) | SALK_077502 (ect3-1) | NASC | N577502, N2110123 | |
| Genetic reagent (A. thaliana) | te234 (ect2-1/ect3-1/ect4-2) | Arribas-Hernández et al., 2018 | N2110132 | Donated to NASC and ABRC |
| Genetic reagent (A. thaliana) | ECT3pro:FLAG-DmADARE488Qcd-ECT3ter | This paper (see Methods) | Seed requests to pbrodersen@bio.ku.dk | |
| Genetic reagent (A. thaliana) | ect3-1/ECT3pro:ECT3-FLAG-DmADARE488Qcd-ECT3ter | This paper (see Methods) | Seed requests to pbrodersen@bio.ku.dk | |
| Genetic reagent (A. thaliana) | te234/ECT3pro:ECT3-FLAG-DmADARE488Qcd-ECT3ter | This paper (see Methods) | Seed requests to pbrodersen@bio.ku.dk | |
| Genetic reagent (A. thaliana) | ect2-1/ECT2pro:ECT2-FLAG-DmADARE488Qcd-ECT2ter | Arribas-Hernández et al., 2021 | ||
| Genetic reagent (A. thaliana) | te234/ECT2pro:ECT2-FLAG-DmADARE488Qcd-ECT2ter | Arribas-Hernández et al., 2021 | ||
| Genetic reagent (A. thaliana) | ect2-1/ECT2pro:ECT2-mCherry-ECT2ter | Arribas-Hernández et al., 2018; Arribas-Hernández et al., 2020 | N2110839, N2110840 | Donated to NASC and ABRC |
| Genetic reagent (A. thaliana) | te234/ECT2pro:ECT2W464A-mCherry-ECT2ter | Arribas-Hernández et al., 2018; Arribas-Hernández et al., 2020 | N2110847, N2110848 | Donated to NASC and ABRC |
| Genetic reagent (A. thaliana) | GFP:WIP1 | Xu et al., 2007 | ||
| Genetic reagent (A. thaliana) | ect2-1/+ GFP: WIP1ECT2pro:ECT2- mCherry-ECT2ter | This paper (see Methods) | Seed requests to pbrodersen@bio.ku.dk | |
| Antibody | anti-mCherry (rabbit polyclonal) | Abcam | ab183628 | Used for WB (1:1000) |
| Chemical compound, drug | Glufosinate-ammonium (PESTANAL) | Sigma | 45520 77182-82-2 | Used for selection of transgenic lines |
| Sequence-based reagent | USER cloning primers | This paper (Appendix) | Used for cloning. Sequences are in the Appendix | |
| Commercial assay or kit | RNeasy Plus Micro kit (inc RLT buffer) | QIAGEN | Cat. # 74,034 | Used for RNA-extraction of FACS-sorted protoplasts |
| Commercial assay or kit | Illumina DNA Nextera Flex kit (now called Illumina DNA Prep) | Illumina | Cat. # 20018704 | Used for Smart-seq2 library preparation |
| Commercial assay or kit | NEBNext small RNA library prep set for Illumina | NEB | Cat. # E7330S | Used for sRNA-seq library preparation |
| Software, algorithm | R | https://www.R-project.org/ | Used for data analyses | |
| Software, algorithm | hyperTRIBER | Rennie et al., 2021; https://github.com/sarah-ku/hyperTRIBER; https://github.com/sarah-ku/targets_arabidopsis | Used for calling significant ADAR-edited sites | |
| Software, algorithm | cutadapt | Kechin et al., 2017 | Used for trimming Smart-seq2, mRNAseq and sRNA-seq reads | |
| Software, algorithm | STAR | Dobin et al., 2013 | Used for mapping Smart-seq2, mRNAseq and sRNA-seq reads | |
| Software, algorithm | nanoPARE and PAS analysis pipelines | Schon et al., 2018; https://github.com/Gregor-Mendel-Institute/nanoPARE; https://github.com/maschon0/ect_polyA_analysis | Used for PAS/PAC analyses | |
| Software, algorithm | bedtools | Quinlan and Hall, 2010 | Used to merge polyadenylation clusters | |
| Software, algorithm | Salmon | Patro et al., 2017 | Used for transcript quantification (Smart-seq2 and mRNAseq) | |
| Software, algorithm | featureCounts | Liao et al., 2014 | Used for sRNA-seq quantification | |
| Software, algorithm | DESeq2 | Love et al., 2014 | Used for DEA (Smart-seq2, mRNAseq and sRNA-seq) | |
| Software, algorithm | gprofiler2 | Raudvere et al., 2019 | Used for GO-term enrichment analysis | |
| Software, algorithm | ggplot2 | https://ggplot2.tidyverse.org | Used to generate plots | |
| Software, algorithm | ImageJ | Schindelin et al., 2012 | Used to generate fluorescence intensity plots | |
| Software, algorithm | IGV (Integrative Genomics Viewer) | Robinson et al., 2011 | Used to navigate and represent genomic data |
Additional files
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Supplementary file 1
ECT3 HyperTRIBE data.
- https://cdn.elifesciences.org/articles/72377/elife-72377-supp1-v2.xlsx
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Supplementary file 2
ECT2 and ECT3 HyperTRIBE data in triple versus single mutants.
- https://cdn.elifesciences.org/articles/72377/elife-72377-supp2-v2.xlsx
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Supplementary file 3
ECT2/ECT3-target sets.
- https://cdn.elifesciences.org/articles/72377/elife-72377-supp3-v2.xlsx
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Supplementary file 4
Poly(A) site selection data.
- https://cdn.elifesciences.org/articles/72377/elife-72377-supp4-v2.xlsx
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Supplementary file 5
Differential expression analysis of ect2/ect3/ect4 mutants (mRNA).
- https://cdn.elifesciences.org/articles/72377/elife-72377-supp5-v2.xlsx
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Supplementary file 6
Differential expression analysis of ect2/ect3/ect4 mutants (miRNAs).
- https://cdn.elifesciences.org/articles/72377/elife-72377-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/72377/elife-72377-transrepform1-v2.pdf
