Completing the TRB family: newly characterized members show ancient evolutionary origins and distinct localization, yet similar interactions

Telomere repeat binding proteins (TRBs) belong to a family of proteins possessing a Myb-like domain which binds to telomeric repeats. Three members of this family (TRB1, TRB2, TRB3) from Arabidopsis thaliana have already been described as associated with terminal telomeric repeats (telomeres) or short interstitial telomeric repeats in gene promoters (telo-boxes). They are also known to interact with several protein complexes: telomerase, Polycomb repressive complex 2 (PRC2) E(z) subunits and the PEAT complex (PWOs-EPCRs-ARIDs-TRBs). Here we characterize two novel members of the TRB family (TRB4 and TRB5). Our wide phylogenetic analyses have shown that TRB proteins evolved in the plant kingdom after the transition to a terrestrial habitat in Streptophyta, and consequently TRBs diversified in seed plants. TRB4-5 share common TRB motifs while differing in several others and seem to have an earlier phylogenetic origin than TRB1-3. Their common Myb-like domains bind long arrays of telomeric repeats in vitro, and we have determined the minimal recognition motif of all TRBs as one telo-box. Our data indicate that despite the distinct localization patterns of TRB1-3 and TRB4-5 in situ, all members of TRB family mutually interact and also bind to telomerase/PRC2/PEAT complexes. Additionally, we have detected novel interactions between TRB4-5 and EMF2 and VRN2, which are Su(z)12 subunits of PRC2. Supplementary Information The online version contains supplementary material available at 10.1007/s11103-023-01348-2.


Supplemental Fig. 2 Conserved residues and electrostatic charge visualization of the Myblike domain in Arabidopsis members of Dicots from TRB_B, TRB_C and TRB_D lineages
The representative members of Dicots from TRB_B, TRB_C and TRB_D lineages, namely A. thaliana TRB4, TRB1 and TRB2, respectively, were analyzed. The three-dimensional model of the Myb-like domains from the site opposing the DNA-binding (A-C) and from the DNA binding (D-F) viewpoints are based on the hTRF2-DNA interaction model (PDB: 1WOU) (Court et al. 2005).
B) and E) The evolutionary dynamics of aa substitutions among aa residues were visualized in ConSurf 2016 (Ashkenazy et al., 2016). The conservation of residues is presented in a scale, where the most conserved residues are shown in dark magenta and non-conserved residues as white.
C) and F) Surface models showing the charge on the Myb-like domains. Residue charges are coded as red for acidic, blue for basic, and white for neutral, visualized using PyMol, Version 2.4.1, Schrödinger, LLC.

Supplemental Fig. 5 Mutual TRB interactions detected by BiFC in N. benthamiana
Protein-protein interactions of TRB proteins fused with nYFP or cYFP part were detected in N. benthamiana leaf epidermal cells by confocal microscopy. Shown here are single images of fluorescence signals from individual channels (YFP, Yellow fluorescence protein; RFP, red fluorescence proteinan internal marker for transformation and expression) and merged signals (merge, merged YFP and RFP channels). Scale bars = 5 µm.

Supplemental Fig. 6 Maximum Intensity projections and Z-stacks of N. benthamiana epidermal cells nuclei presenting BiFC analyses
Maximum Intensity Projections (I) of entire Z-stack images (II) of nuclei of N. benthamiana leaf epidermal cells displaying BiFC interactions of TRB proteins. Shown here are merged images of YFP (interaction of the tested proteins) and mRFP (internal marker for transformation and expression) fluorescence detected by confocal microscopy. Scale bars = 5 μm.

Supplemental Fig. 7 Interactions of TRB4-5 with TERT domains
A) Schematic depiction of the plant catalytic subunit of telomerase (TERT) showing functional motifs. The regions of structural domains TEN (telomerase essential Nterminal domain), TRBD (Telomerase RNA-binding domain), RT (reverse transcriptase domain) and CTE (C-terminal extension) are depicted above the conserved RT motifs (1, 2, A, B, C, D and E), telomerase-specific motifs (T2, CP, QFP and T) and a NLS (nucleus localization-like signal). All of the depicted TERT fragments were used for proteinprotein interaction analysis (amino acid numbering is shown). B) TERT fragments from Majerská et al. 2017 were fused with the GAL4 DNA-binding domain (BD). TRB4 and TRB5 were fused with the GAL4 activation domain (AD). Both constructs were introduced into yeast strain PJ69-4a carrying reporter genes His3 and Ade2. Interactions were detected on histidine-deficient SD medium (-His), or under stringent adenine-deficient SD medium (-Ade) selection. Co-transformation with an empty vector (AD, BD) served as a negative control. Asterisks *, 3 mM 3-aminotriazol.