Abstract
Key message
Duplicate POT1 genes must rapidly diverge or be inactivated.
Abstract
Protection of telomeres 1 (POT1) encodes a conserved telomere binding protein implicated in both chromosome end protection and telomere length maintenance. Most organisms harbor a single POT1 gene, but in the few lineages where the POT1 family has expanded, the duplicate genes have diversified. Arabidopsis thaliana bears three POT1-like loci, POT1a, POT1b and POT1c. POT1a retains the ancestral function of telomerase regulation, while POT1b is implicated in chromosome end protection. Here we examine the function and evolution of the third POT1 paralog, POT1c. POT1c is a new gene, unique to A. thaliana, and was derived from a duplication event involving the POT1a locus and a neighboring gene encoding ribosomal protein S17. The duplicate S17 locus (dS17) is highly conserved across A. thaliana accessions, while POT1c is highly divergent, harboring multiple deletions within the gene body and two transposable elements within the promoter. The POT1c locus is transcribed at very low to non-detectable levels under standard growth conditions. In addition, no discernable molecular or developmental defects are associated with plants bearing a CRISPR mutation in the POT1c locus. However, forced expression of POT1c leads to decreased telomerase enzyme activity and shortened telomeres. Evolutionary reconstruction indicates that transposons invaded the POT1c promoter soon after the locus was formed, permanently silencing the gene. Altogether, these findings argue that POT1 dosage is critically important for viability and duplicate gene copies are retained only upon functional divergence.
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Acknowledgements
The authors thank Holger Puchta for sharing plasmids and expertise on CRISPR, Dan Browne for assistance with computational analysis, and members of the Shippen Lab for critical feedback on the project.
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This work was supported by a grant from the National Institutes of Health (GM065383 to D.E.S.).
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Fig. S1
Transcriptional analysis of POT1c. a Results from RT-PCR analysis of POT1c using nested PCR for flowers. Left: first PCR (40 cycles), and right: second (nested) PCR (40 cycles). b Results from RT-PCR analysis of POT1c using nested PCR for flowers, seedlings, and leaves. Individual bands were gel purified, and cloned into the pGEM-T vector for sequencing. The most prominent band (closed triangle) in each reaction contained a 717 nucleotide sequence corresponding to fully spliced POT1c (GenBank Accession MK585073). A few other bands (open triangles) displayed evidence of incomplete splicing or consisted of unrelated sequence, indicating they were non-specific amplification products. Fig. S2 Generation of a POT1c mutant. a Location of the deletion by CRISPR-Cas9 is indicated by the black triangle. Location of the internal ATG is indicated by the red arrow. b Chromatogram of WT, mutant (Mt), and heterozygous (Het) plants at the POT1c locus. Vertical line indicates the start of deletion. Heterozygous chromatograms containing forward sequence (Het Fw) and reverse sequence (Het Rv) are shown. Fig. S3 The pot1c-1 mutant does not exhibit a defect in telomere maintenance or telomerase activity. a Bulk telomere length of WT plants and plants deficient in either POT1a or POT1c. b Telomere length on the individual chromosome arms 1L or 3R in WT plants and plants deficient in either POT1a or POT1c. No difference in telomere length was observed. c Telomerase activity levels were compared for WT, pot1a (p = 0.0007), and pot1c (p = 0.81) mutants using quantitative TRAP. N = 4. *p < 0.05.Fig. S4 The POT1c transcript is not actively silenced. a Results for Chop PCR. No DNA (lane 1), untreated (lane 2), treated with DNA methylation-sensitive restriction enzymes SnaBI, HypCH4IV, and HhaI (lane 3), or treated with the restriction enzyme McrBC that cuts at sites of CG methylation (lane 4). Red asterisks indicate amplification of DNA from incomplete digestion by McrBC. b qPCR results for POT1c and POT1a transcripts in ddm1 and dcl2,3,4 mutants. POT1a, POT1c, and TAS1 transcripts were normalized to the GAPDH transcript level. The fold change in POT1c transcript was adjusted relative to POT1a. The mir173 target TAS1 was used as a control in the dcl2,3,4 mutant. c Results of RT-PCR in the ddm1. PCR data for WT and ddm1 were compared to the AtMu1 TSA2 transposable element to confirm the ddm1 mutation. Fig. S5 The POT1c locus lacks a functional promoter. a Positive control: GUS reporter under the control of the CYCB1 promoter in a pot1a mutant background. Negative control: untransformed WT. GUS reporter expressed from the POT1c promoter. SAM: shoot apical meristem. b PCR amplification of the POT1cp-GUS construct in three transformants. PCR amplification of the ATR locus is shown at the bottom for DNA quality. Fig. S6 Original figure of Fig. 6b. Forced expression of POT1c leads to perturbations in telomere length maintenance and decreased telomerase activity. Results of TRF analysis to measure bulk telomere length of wild type (WT) and POT1cOE plants. Table S1 PCR primer sequences. Table S2 Accessions used in Fig. 4b. (PDF 15211 kb)
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Kobayashi, C.R., Castillo-González, C., Survotseva, Y. et al. Recent emergence and extinction of the protection of telomeres 1c gene in Arabidopsis thaliana. Plant Cell Rep 38, 1081–1097 (2019). https://doi.org/10.1007/s00299-019-02427-9
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DOI: https://doi.org/10.1007/s00299-019-02427-9