Nucleic Acids Research

This study identifies a novel operon-driven signaling module in Deinococcus radiodurans. The operon includes a von Willebrand A domain protein (DRA0331), a Ser/Thr protein kinase (DRA0332), a canonical FHA-domain protein (DRA0333), and a PP2C-type phosphatase (DRA0334). DRA0334 shows Mn{superscript 2}{square}-dependent phosphatase activity and has a unique dual-domain structure that combines a Kinase-Interacting FHA (KI-FHA) domain with a PP2C catalytic domain. Functional assays show that FHA-domain protein, DRA0333 boosts the phosphorylation of STPKs like RqkA and DR1243 while operonic partner PP2C-type phosphatase, DRA0334 counteracts this through targeted dephosphorylation, establishing a phospho-regulatory antagonism. Notably, the KI-FHA domain of the DRA0334 phosphatase competitively interacts with the FHA domain to modulate the radiation-responsive RqkA kinase, thereby maintaining kinase-phosphatase balance. This KI-FHA domain also imparts substrate specificity and enables feedback regulation. Additionally, DRA0334 modular variants confirm separate roles of catalytic and docking modules, and STRING analyses link DRA0334 functions to DNA repair and stress recovery. Collectively, the findings suggest an operonic connection between DRA0333 and DRA0334, indicating that the KI-FHA and FHA domains may act as phospho-docking switches. These switches can regulate both kinase and phosphatase activities in a push-pull regulatory mechanism within the phosphorylation-dephosphorylation cycle of signal transduction, depending on their association with the type of catalytic domain.

The Rev1 deoxycytidyl transferase functions as a scaffold protein for DNA polymerase {zeta} (Pol {zeta})-mediated translesion synthesis (TLS). Biochemical studies with yeast enzymes indicate that Rev1 plays a dual regulatory role in TLS, stimulating Pol {zeta} activity at sites of damage but inhibiting its activity on undamaged DNA. An evolutionary conserved N-terminal alpha-helical motif (M1), located 10-20 amino acids upstream of Rev1s single BRCT domain, is required for the inhibitory activity of Rev1 on undamaged DNA. Mutations in the M1 motif result in a stimulation of Pol {zeta} replication activity on both undamaged and damaged DNA. Yeast cells carrying a REV1 mutant lacking the M1 motif, show a significant increase in mutation track length, without significantly affecting overall spontaneous mutation rates. The regulatory activity of Rev1 is independent of its catalytic activity. However, it requires that Rev1-Pol {zeta} is a stable complex, and that this complex is coordinated by the replication clamp PCNA. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/700666v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@9b40dorg.highwire.dtl.DTLVardef@10bf5d1org.highwire.dtl.DTLVardef@376ff4org.highwire.dtl.DTLVardef@1970db8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Time-resolved transcriptomic profiling has been used to study phage-host interactions for more than a decade. However, the resulting datasets are not readily accessible for custom re-analysis, and resources are lacking that provide standardized processing, storage, and analysis of transcriptomes from phage infections. Here, we present the PhageExpressionAtlas, the first bioinformatics resource for storing time-resolved dual RNA-sequencing data from phage infections. This data was processed uniformly using a custom analysis pipeline and is presented for interactive exploration through visualisation. The PhageExpressionAtlas currently hosts 42 datasets from 23 studies. Using the PhageExpressionAtlas, we replicate key findings from original publications and extend hypothesis testing across multiple phage-host systems. By systematically querying and analyzing the underlying database, we evaluate approaches to phage gene classification and show that uncharacterized phage genes are expressed across all infection phases. Moreover, we provide a comprehensive view of the expression dynamics of anti-phage defenses as well as host- and phage-encoded anti-defense systems in the infection context, indicating unique and conserved patterns of transcriptional regulation underlying bacterial anti-phage immunity and phage counter-strategies. Together, the PhageExpressionAtlas is a unifying resource that democratizes transcriptomics-driven analyses of phage-host interactions and supports integrative cross-study assessment.

Mammalian telomeric DNA comprises long tracts of tandem TTAGGG repeats. The same repeats are also found at internal chromosomal regions called interstitial telomeric sequences (ITSs). Telomeres are transcribed into UUAGGG-containing transcripts, named TERRA, which serve multiple functions in maintaining telomere integrity. Complementary RNAs containing C-rich telomeric repeats, named ARIA, have also been identified in few yeast mutants and mammalian cells with dysfunctional telomeres. The molecular features and functions of ARIA remain understudied, mainly due to its low abundance and the lack of suitable cellular systems. Here, we show that Chinese hamster ovary (CHO) cells produce abundant TERRA and ARIA transcripts, predominantly originating from ITSs. Both RNAs are polyadenylated, exhibit relatively short half-lives and form large cellular foci. We also show that ARIA depletion leads to exposure of single-stranded (ss) DNA at ITSs and that ssDNA exposure increases when ITS DNA is damaged. SsDNA formation does not require the DNA damage signaling kinases ATM and ATR, nor the exonucleases DNA2 and EXO1; however, ATM prevents excessive ssDNA accumulation when ARIA function is inhibited. These findings establish CHO cells as a powerful model to dissect telomeric RNA functions and reveal ARIA as a key regulator of telomeric repeat DNA integrity.

Telomeres are nucleoprotein structures at the ends of eukaryotic chromosomes that safeguard them from triggering inappropriate DNA damage signaling. POT1, a member of the mammalian shelterin complex, binds single-stranded (ss) telomeric DNA and blocks the activation of the ATR kinase-mediated DNA damage response at telomeres. Yet until recently, it was poorly understood how the double-stranded (ds)-ss telomeric junction was protected from DNA damage response factors. An initial study of the DNA-binding activity of human POT1 (hPOT1) using systematic evolution of ligands by exponential enrichment (SELEX) and subsequent investigation revealed that POT1 contains a binding pocket, known as the POT-hole, that binds the 5 phosphorylated dC of the telomeric ds-ss junction. The amino acid residues composing the POT-hole show full sequence identity with telomeric proteins from diverse eukaryotes, including Caenorhabditis elegans POT-1. The current study builds on this SELEX method, developing an extensive analysis pipeline for SELEX datasets sequenced by next-generation sequencing and achieving a deeper analysis of the resulting sequences. We validated our approach by applying it to the DNA-binding domain of hPOT1, yielding results consistent with a previous SELEX study. Furthermore, we employ our pipeline to characterize the DNA-binding activity of C. elegans proteins that are considered homologs of hPOT1: POT-1, POT-2, POT-3, and MRT-1. Our analysis suggests that all four proteins show a binding preference for G-enriched DNA sequences, with POT-1 additionally binding secondary structural elements. Overall, we present a bioinformatics pipeline that is accessible and applicable for determining the nucleic acid-binding properties of a variety of proteins.

The hyper-modified DNA base J helps control termination of Pol II transcription at polycistronic transcription units (PTUs) in T. brucei and L. major, allowing epigenetic control of gene expression. The Telomere Repeat-containing RNA (TERRA) is synthesized in T. brucei by Pol I readthrough transcription of a telomeric PTU. While little is understood regarding TERRA synthesis and function, the hyper-modified DNA base J is highly enriched at telomeres in L. major promastigotes. We now show that TERRA is synthesized by Pol II in L. major and loss of base J leads to increased TERRA from multiple telomeric ends, presumably via readthrough transcription from an adjacent PTU. Furthermore, Pol II readthrough defects and increased TERRA correlate with increased telomeric DNA damage, increased differentiation of promastigotes to the infectious metacyclic life stage and decreased cell viability. These results help explain the essential nature of base J in Leishmania and provide insight regarding epigenetic control of RNA expression, telomere stability and parasite development during the life cycle of L. major.

RNApdbee 3.0 (publicly available at https://rnapdbee.cs.put.poznan.pl/) offers an advanced pipeline for comprehensive RNA structural annotation, integrating 2D and 3D data to build detailed nucleotide interaction networks. It classifies base pairs as canonical or noncanonical using the Leontis-Westhof and Saenger schemes and identifies stacking, base-ribose, base-phosphate, and base-triple interactions. The tool handles incomplete or modified residues, marking missing nucleotides and distinguishing noncanonical base pairs for accurate and effective visualization. Results are provided in standard formats - namely, extended dot-bracket notation, BPSEQ, and CT - and in detailed graphical visualizations. RNApdbee decomposes 2D structures into stems, loops, and single strands and offers flexible pseudoknot encoding. Its unified framework addresses inconsistencies across structural data formats by standardizing all inputs to PDBx/mmCIF and integrating seven widely used annotation tools. Finally, RNApdbee ensures reliable, format-independent, and comprehensive RNA structural annotation and interpretation.

Post-transcriptional gene regulation is a key mechanism for bacterial stress responses and virulence, and RNA-mediated regulation frequently relies on global RNA-binding proteins (RBPs). A pair of interacting KH-domain proteins (KhpA and KhpB) have recently been identified as global RBPs in several bacterial species. To better understand their molecular functions, we employed bacterial two- and three-hybrid (B2H/B3H) assays in an E. coli reporter system to analyze protein-protein and protein-RNA interactions of KhpA/B orthologs from three human pathogens: Campylobacter jejuni, Helicobacter pylori, and Clostridioides difficile. Protein-protein interactions were conserved across all species, with KhpA-KhpB heterodimers forming more robustly than either homodimer and KhpA homodimerizing more readily than KhpB. On the other hand, protein-RNA interactions were more varied across species: C. jejuni and C. difficile KhpA bound both species-specific and non-specific RNAs, but H. pylori KhpA--and KhpB orthologs from all species--showed no RNA interaction in B3H assays. Site-directed mutagenesis experiments demonstrated that residues in the GXXG motif of KhpA are critical for RNA interaction and differences in these residues account for the distinct RNA-binding behaviors of KhpA orthologs. Collectively, these findings provide a cross-species, molecular view of how KhpA and KhpB recognize one another and RNA ligands to regulate gene expression.

RNA structure plays a crucial role in diverse biological processes beyond the translation of genetic information. Therefore, the development of reliable methods for RNA structure prediction is essential for understanding RNA structure-related functions, however accurate and comprehensive RNA structure prediction remains challenging. Here, we focus on secondary structure prediction of transfer RNA (tRNA) using structure probing coupled with next-generation sequencing (tRNA Structure-seq). In silico prediction of Saccharomyces cerevisiae tRNA secondary structures achieves only 56.9% accuracy on average. Incorporation of dimethyl sulfate (DMS) probing data improve prediction accuracy to 87.4%, which is still not sufficient for practical tRNA structure prediction. To overcome this, we optimized the tRNA Structure-seq analysis pipeline by explicitly incorporating natural tRNA modifications detected in tRNA sequencing data and by refining pseudo-free energy parameters specifically optimized for tRNA structure prediction. Using this optimized pipeline, the average prediction accuracy is remarkably improved to 94%. Furthermore, analysis of multiple structural conformations predicted from DMS probing data indicates that S. cerevisiae tRNAs predominantly adopt the canonical cloverleaf secondary structure under in vivo conditions. Finally, we examined tRNA structures under mild stress conditions, including heat stress, osmotic stress, and antibiotic stress. These perturbations had minimal effects on in vivo tRNA secondary structure, demonstrating that S. cerevisiae tRNAs maintain structural stability under physiologically relevant stress conditions. In summary, our results establish an optimized tRNA Structure-seq analysis that enables highly accurate tRNA secondary structure prediction and reveals the intrinsic robustness of tRNA structures in living cells.

Eukaryotic cells regulate the assembly and activation of the essential DNA helicase at the heart of the chromosome replication machinery, to ensure that the chromosomes are copied just once per cell cycle. The Mcm10 protein is essential for helicase activation in budding yeast, but an equivalent role for MCM10 orthologues in animal cells has not been explored. Moreover, complete deletion of the mcm-10 gene is viable in the nematode Caenorhabditis elegans, suggesting the involvement of additional factors. Here we show that MCM-10 and a second factor called SLD-2 are recruited to chromatin after helicase assembly in the C. elegans early embryo and are jointly required for helicase activation. Moreover, deletion of the Mcm10 gene is viable in mouse embryonic stem cells, but causes synthetic lethality in the absence of RECQL4, which is the orthologue of SLD-2 in vertebrate species. Helicase activation is blocked in the combined absence of MCM10 and RECQL4, mirroring the situation in C. elegans. These findings indicate that metazoan helicase activation requires two conserved factors that are mutated in human disease syndromes.

RNA-binding proteins (RBPs) regulate essential aspects of RNA metabolism, yet accurately identifying RNA-binding domains (RBDs) and quantifying the impact of sequence variation on RNA-binding ability remain challenging. Here, we present HERCULES (Hybrid framEwoRk for RNA-binding domain loCalization and mUtation anaLysis using physicochemical and languagE modelS), a unified sequence-based framework for simultaneous RBD localization, global RNA-binding propensity prediction and mutation effect assessment. HERCULES integrates a fine-tuned protein language model with an explicit residue-level physicochemical module, combining global contextual representations with local mutation-sensitive descriptors. On an independent test set, the HERCULES global score discriminates RBPs from non-RBPs with an AUROC of 0.86. At residue resolution, HERCULES outperforms state-of-the-art sequence-based predictors in identifying canonical, non-canonical and putative RBDs across Pfam-annotated proteins. Using a curated dataset of experimentally validated RNA-binding-disrupting mutations, HERCULES correctly classifies 87% of deleterious variants, including single-amino acid substitutions. Evaluation on experimentally resolved protein-RNA complexes further demonstrates robust residue-level performance and improved generalization when contact annotations are augmented with AlphaFold3-predicted complexes. By unifying domain localization and mutation sensitivity within a single sequence-only framework, HERCULES provides a mechanistically interpretable approach for studying RNA-protein interactions. HERCULES is freely available at https://tools.tartaglialab.com/hercules and as an open-source Python package at https://github.com/tartaglialabIIT/hercules.git.

N4-acetylcytidine (ac4C) is a conserved RNA modification that enhances RNA stability and translation accuracy. Emerging evidence suggests that ac4C levels can change in response to cellular and environmental triggers. Despite these indications of regulatory dynamics, the enzymes responsible for removing ac4C, beyond the recently proposed rRNA deacetylase SIRT7, remain largely unknown. Here, we examined 19 ASCH domain-containing proteins from bacteria, archaea, and humans to determine their possible activity in tRNA deacetylation. Despite differences in their sequences, structures, and nucleic-acid binding properties, all tested proteins were capable of removing ac4C from tRNA, revealing a conserved deacetylase activity across diverse species. The proteins were found to vary in nucleic acid recognition, including an archaeal specific helix-turn-helix domain that promotes strong tRNA binding. Together, these findings establish ASCH proteins as a widespread and previously unrecognized family of tRNA deacetylases, suggesting that enzymatic ac4C turnover may require complex regulation within the cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/708502v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@880691org.highwire.dtl.DTLVardef@674a13org.highwire.dtl.DTLVardef@1304045org.highwire.dtl.DTLVardef@cba7f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

MutY excises adenine (A) from 8-oxo-guanine:adenine (OG:A) lesions in DNA to initiate base excision repair (BER) and thereby prevent mutations. A catalytic Glu, found at position 43 in the enzyme from Geobacillus stearothermophilus (Gs MutY), protonates the nucleobase at N7 to labilize the N-glycosidic bond. The resulting oxocarbenium ion transition state is stabilized by a covalent DNA-enzyme intermediate and resolved by nucleophilic attack to yield the beta-anomer abasic AP site product. The retaining SN1 mechanism for MutY posits deprotonation of the nucleophile by the catalytic Glu. Here we tested kinetic and structural consequences of Glu replacement and found that E43Q and E43S substitution variants were severely impaired, retained measurable activity, but engage the substrate nucleobase in an anti conformation, rotated by 180 {degrees} from the syn conformation seen in previous substrate complexes. The enzyme-generated AP product is observed in its alpha-anomer configuration for these Glu-replacement variants. Comparison with inverting adenine glycosylases that act on RNA or nucleosides shows that MutYs mechanism is uniquely reliant on one catalytic residue for both leaving group and nucleophile activation, a situation that may serve to ensure only rare adenines paired with OG are excised.

The ability to access, search, and analyse large collections of RNA molecules together with their secondary structure and evolutionary context is essential for comparative and phylogeny-driven studies. Although RNA secondary structure is known to be more conserved than primary sequence, no existing resource systematically associates individual RNA molecules with curated phylogenetic classifications. Here, we introduce PhyloRNA, a curated meta-database that provides large-scale access to RNA secondary structures collected from public resources or derived from experimentally resolved 3D structures. PhyloRNA allows users to search, select, and download extensive sets of RNA molecules in multiple textual formats, each entry being explicitly linked to phylogenetic annotations derived from five curated taxonomy systems. In addition to taxonomic information, each RNA molecule is accompanied by a rich set of descriptors, including pseudoknot order, genus, and three levels of structural abstraction--Core, Core Plus, and Shape--which facilitate comparative analyses across sets of molecules. PhyloRNA is publicly available at https://bdslab.unicam.it/phylorna/ and is regularly updated to incorporate newly available data and revised taxonomic annotations.

Small regulatory RNAs (sRNAs) are major post-transcriptional regulators in bacteria and, together with transcriptional regulators such as the two-component systems (TCSs), participate in the rapid adaptation of these microorganisms to changing environments. Several examples of paralogous sRNAs with overlapping functions have been reported, that could in theory integrate different environmental cues. Consistent with this idea, we have identified the acid-responsive RstB-RstA two-component system, important for virulence of multiple bacterial species, as a specific multicopy activator of the Escherichia coli OmrB sRNA, but not of the paralogous sRNA OmrA. Further characterization of this regulation unexpectedly revealed the asr-ydgU operon, itself a target of RstB-RstA, as a dual modulator of this TCS via two opposite effects. First, the 27 aminoacids YdgU small protein exerts a negative feedback by directly interacting with RstB and, second, Asr in contrast mediates a positive feedback on RstB-RstA activity via a not completely elucidated mechanism. These results provide a new example of retro-control of a TCS, here RstB-RstA, by one of its direct targets. They further highlight the major role of small proteins in controlling TCS activity and ydgU was thus renamed samT, for Small Acid-responsive Modulator of the RstB-RstA TCS.

Artificial microRNAs (amiRNAs) offer a powerful strategy for targeted gene silencing, but their rational design is limited by complex sequence-structure-processing relationships and the lack of tools capable of optimizing efficacy and specificity. To address this need, we developed miRarchitect, a web-based platform that uses machine learning to support the customizable design of amiRNAs. miRarchitect integrates neural network-guided target-site selection, siRNA insert design, and scaffold choice, utilizing large-scale data from human primary microRNAs (pri-miRNAs) and next-generation sequencing. The platform generates molecules that closely resemble endogenous pri-miRNAs and includes comprehensive off-target analysis to enhance specificity. Experimental validation targeting TMPRSS2 and ACE-2 confirmed precise processing, robust knockdown, and high specificity of miRarchitect-designed amiRNAs. In comparative benchmarking, miRarchitect consistently produced functional amiRNAs, whereas only half of the top candidates generated by other tools showed measurable activity. miRarchitect is freely available at https://rnadrug.ichb.pl/mirarchitect and provides an intuitive interface with an automated workflow for generating, ranking, and selecting candidate amiRNAs for research and therapeutic applications.

The XerC and XerD recombinases function with accessory proteins PepA, ArcA and ArgR at plasmid recombination sites such as psi and cer, to keep multicopy plasmids in a monomeric state and ensure stable plasmid inheritance. Xer recombination acts on plasmid recombination sites only if they are directly repeated and recombination produces a specific catenane in which the two product circles are interlinked around each other exactly four times. Here we measure the precise change in topological linkage ({Delta}Lk) that occurs during Xer recombination at psi. We use a DNA substrate with close-spaced psi sites that recombines to produce one circle of 398 bp and another of 3039 bp and demonstrate that the small circle is exclusively the -1 topoisomer. Using a purified topoisomer of the substrate, we show that Xer recombination proceeds with a linkage change ({Delta}Lk) of +4. Similar experiments using a substrate with equally spaced psi sites agreed with this result. The measured linkage change is consistent with a reaction mechanism for tyrosine recombinases in which the sites align antiparallel prior to recombination and recombine via a Holliday junction intermediate. Four negative supercoils are converted to catenation nodes by strand exchange, providing an energetic driving force for the reaction. We compare this to the mechanism of serine recombinases and the recently discovered bridge RNA-guided recombinases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/706985v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1fc8038org.highwire.dtl.DTLVardef@460174org.highwire.dtl.DTLVardef@9700borg.highwire.dtl.DTLVardef@19afbd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

APOBEC3A catalyzes cytosine-to-uracil deamination in single-stranded DNA and RNA. Physiologically, APOBEC3A functions in innate immunity and aberrant deamination is associated with cytosine mutations in enzymatically preferred YTCW substrate motifs in multiple cancers. Much less is known about the potential contribution of APOBEC3A-catalyzed RNA editing to virus and cancer evolution. Here, we present HAMMER (hairpin-based APOBEC3A-mediated mRNA editing reporter), a rapid luminescence-based cellular assay for measuring RNA editing by APOBEC3A. HAMMER reports APOBEC3A activity as a reduction in the ratio of firefly to renilla luciferase activity. Briefly, tandem renilla and firefly luciferase open reading frames are separated by an optimal APOBEC3A hairpin substrate, in which C-to-U editing of a CGA motif yields a UGA stop codon thus preventing translation of the downstream firefly luciferase reporter, without impacting the upstream renilla reporter. HAMMER activation is dose-responsive, catalytic activity-dependent, and specific to human APOBEC3A. A panel of herpesviral ribonucleotide reductase constructs was used to show that direct inhibition of APOBEC3A results in a dose-responsive recovery of firefly luciferase expression. HAMMER is therefore a scalable and easy-to-use method for quantifying cellular APOBEC3A RNA editing activity and characterizing inhibitors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/695965v2_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@11b1f87org.highwire.dtl.DTLVardef@1b3110dorg.highwire.dtl.DTLVardef@1249c8aorg.highwire.dtl.DTLVardef@a133e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

The RNA exosome is an essential and ubiquitous RNase with exonucleolytic activity, involved in ribosome biogenesis and RNA quality control in eukaryotes. It is present both in nucleus and cytoplasm, and interacts with specific cofactors in each cell compartment, which are essential for recruitment and activity control of the exosome. Posttranslational modifications are known to regulate enzyme activity and protein interaction, although their precise roles are individually specific. In this study, we investigated the phosphorylation status of proteins associated with the nuclear (Rrp6) and core (Rrp46) subunits of the RNA exosome in Saccharomyces cerevisiae. Using co-immunoprecipitation followed by phosphopeptide enrichment and high-resolution mass spectrometry, we identified 121 phosphorylation sites on proteins functionally related to rRNA processing. Differential phosphorylation patterns between Rrp6 and Rrp46 co-immunoprecipitations are consistent with distinct exosome assemblies and suggest potential regulatory roles for phosphorylation. The results shown here highlight the role of phosphorylation in the recruitment and control of the exosome in RNA processing and degradation, offering new insights into the posttranscriptional control of gene expression.

Telomere protection and maintenance are mediated by proteins that bind telomeric DNA and recruit additional components of telomeric chromatin. While these factors are well characterized in yeast and mammals, their counterparts in plants remain poorly defined. Here, we used a proteomic approach in Arabidopsis thaliana to identify nuclear proteins that preferentially associate with telomeric DNA. We identified TRFL7, a previously uncharacterized member of the TRF-like (TRFL) protein family, as a prominent candidate. We show that TRFL7, together with its close homologues TRFL5 and TRFL11, associates with telomeric chromatin and forms distinct nuclear foci that preferentially localize near the nucleolus, resembling the nucleolus-associated telomere clustering characteristic of Arabidopsis. Genetic inactivation of TRFL7 in combination with either TRFL5 or TRFL11 results in telomere elongation, indicating a role for these proteins in telomere length homeostasis. Notably, TRFL7 contains an iDDR sequence motif that is also present in human TRF2, where it limits the activity of the Mre11-Rad50-Nbs1 complex. Together, our findings identify TRFL7 as a functional component of plant telomeric chromatin and suggest that it represents a plant orthologue of human TRF2.