RUVBL proteins are involved in plant gametophyte development.

The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA+ family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis.

Optimized Detection of Protein-Protein and Protein-DNA Interactions, with Particular Application to Plant Telomeres.

Characterization of protein-protein and protein-DNA interactions is critical to understand mechanisms governing the biology of cells. Here we describe optimized methods and their mutual combinations for this purpose: bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP), yeast two-hybrid systems (Y2H), and chromatin immunoprecipitation (ChIP). These improved protocols  detect trimeric complexes in which two proteins of interest interact indirectly via a protein sandwiched between them. They also allow isolation of low-abundance chromatin proteins and confirmation that proteins of interest are associated with specific DNA sequences, for example telomeric tracts. Here we describe these methods and their application to map interactions of several telomere- and telomerase-associated proteins and to purify a sufficient amount of chromatin from Arabidopsis thaliana for further investigations (e.g., next-generation sequencing, hybridization).

The plant Pontin and Reptin homologues, RuvBL1 and RuvBL2a, colocalize with TERT and TRB proteins in vivo, and participate in telomerase biogenesis.

Telomerase maturation and recruitment to telomeres is regulated by several telomerase- and telomere-associated proteins. Among a number of proteins, human Pontin and Reptin play critical roles in telomerase biogenesis. Here we characterized plant orthologues of Pontin and Reptin, RuvBL1 and RuvBL2a, respectively, and show association of Arabidopsis thaliana RuvBL1 (AtRuvBL1) with the catalytic subunit of telomerase (AtTERT) in the nucleolus in vivo. In contrast to mammals, interactions between AtTERT and AtRuvBL proteins in A. thaliana are not direct and they are rather mediated by one of the Arabidopsis thaliana Telomere Repeat Binding (AtTRB) proteins. We further show that plant orthologue of dyskerin, named AtCBF5, is indirectly associated with AtTRB proteins but not with the AtRuvBL proteins in the plant nucleus/nucleolus, and interacts with the Protection of telomere 1 (AtPOT1a) in the nucleolus or cytoplasmic foci. Our genome-wide phylogenetic analyses identify orthologues in RuvBL protein family within the plant kingdom. Dysfunction of AtRuvBL genes in heterozygous T-DNA insertion A. thaliana mutants results in reduced telomerase activity and indicate the involvement of AtRuvBL in plant telomerase biogenesis.

Correction to: Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo.

In the published online version, the affiliations were mixed up. Corrected affiliation section is shown below. Also, the update has also been reflected in the author group section above.

Tandem affinity purification of AtTERT reveals putative interaction partners of plant telomerase in vivo.

The life cycle of telomerase involves dynamic and complex interactions between proteins within multiple macromolecular networks. Elucidation of these associations is a key to understanding the regulation of telomerase under diverse physiological and pathological conditions from telomerase biogenesis, through telomere recruitment and elongation, to its non-canonical activities outside of telomeres. We used tandem affinity purification coupled to mass spectrometry to build an interactome of the telomerase catalytic subunit AtTERT, using Arabidopsis thaliana suspension cultures. We then examined interactions occurring at the AtTERT N-terminus, which is thought to fold into a discrete domain connected to the rest of the molecule via a flexible linker. Bioinformatic analyses revealed that interaction partners of AtTERT have a range of molecular functions, a subset of which is specific to the network around its N-terminus. A significant number of proteins co-purifying with the N-terminal constructs have been implicated in cell cycle and developmental processes, as would be expected of bona fide regulatory interactions and we have confirmed experimentally the direct nature of selected interactions. To examine AtTERT protein-protein interactions from another perspective, we also analysed AtTERT interdomain contacts to test potential dimerization of AtTERT. In total, our results provide an insight into the composition and architecture of the plant telomerase complex and this will aid in delineating molecular mechanisms of telomerase functions.

Identification of Nucleolus-Associated Chromatin Domains Reveals a Role for the Nucleolus in 3D Organization of the A. thaliana Genome.

The nucleolus is the site of rRNA gene transcription, rRNA processing, and ribosome biogenesis. However, the nucleolus also plays additional roles in the cell. We isolated nucleoli using fluorescence-activated cell sorting (FACS) and identified nucleolus-associated chromatin domains (NADs) by deep sequencing, comparing wild-type plants and null mutants for the nucleolar protein NUCLEOLIN 1 (NUC1). NADs are primarily genomic regions with heterochromatic signatures and include transposable elements (TEs), sub-telomeric regions, and mostly inactive protein-coding genes. However, NADs also include active rRNA genes and the entire short arm of chromosome 4 adjacent to them. In nuc1 null mutants, which alter rRNA gene expression and overall nucleolar structure, NADs are altered, telomere association with the nucleolus is decreased, and telomeres become shorter. Collectively, our studies reveal roles for NUC1 and the nucleolus in the spatial organization of chromosomes as well as telomere maintenance.

Telomere- and Telomerase-Associated Proteins and Their Functions in the Plant Cell.

Telomeres, as physical ends of linear chromosomes, are targets of a number of specific proteins, including primarily telomerase reverse transcriptase. Access of proteins to the telomere may be affected by a number of diverse factors, e.g., protein interaction partners, local DNA or chromatin structures, subcellular localization/trafficking, or simply protein modification. Knowledge of composition of the functional nucleoprotein complex of plant telomeres is only fragmentary. Moreover, the plant telomeric repeat binding proteins that were characterized recently appear to also be involved in non-telomeric processes, e.g., ribosome biogenesis. This interesting finding was not totally unexpected since non-telomeric functions of yeast or animal telomeric proteins, as well as of telomerase subunits, have been reported for almost a decade. Here we summarize known facts about the architecture of plant telomeres and compare them with the well-described composition of telomeres in other organisms.

Telomere binding protein TRB1 is associated with promoters of translation machinery genes in vivo.

Recently we characterised TRB1, a protein from a single-myb-histone family, as a structural and functional component of telomeres in Arabidopsis thaliana. TRB proteins, besides their ability to bind specifically to telomeric DNA using their N-terminally positioned myb-like domain of the same type as in human shelterin proteins TRF1 or TRF2, also possess a histone-like domain which is involved in protein-protein interactions e.g., with POT1b. Here we set out to investigate the genome-wide localization pattern of TRB1 to reveal its preferential sites of binding to chromatin in vivo and its potential functional roles in the genome-wide context. Our results demonstrate that TRB1 is preferentially associated with promoter regions of genes involved in ribosome biogenesis, in addition to its roles at telomeres. This preference coincides with the frequent occurrence of telobox motifs in the upstream regions of genes in this category, but it is not restricted to the presence of a telobox. We conclude that TRB1 shows a specific genome-wide distribution pattern which suggests its role in regulation of genes involved in biogenesis of the translational machinery, in addition to its preferential telomeric localization.

Telomere repeat binding proteins are functional components of Arabidopsis telomeres and interact with telomerase.

Although telomere-binding proteins constitute an essential part of telomeres, in vivo data indicating the existence of a structure similar to mammalian shelterin complex in plants are limited. Partial characterization of a number of candidate proteins has not identified true components of plant shelterin or elucidated their functional mechanisms. Telomere repeat binding (TRB) proteins from Arabidopsis thaliana bind plant telomeric repeats through a Myb domain of the telobox type in vitro, and have been shown to interact with POT1b (Protection of telomeres 1). Here we demonstrate co-localization of TRB1 protein with telomeres in situ using fluorescence microscopy, as well as in vivo interaction using chromatin immunoprecipitation. Classification of the TRB1 protein as a component of plant telomeres is further confirmed by the observation of shortening of telomeres in knockout mutants of the trb1 gene. Moreover, TRB proteins physically interact with plant telomerase catalytic subunits. These findings integrate TRB proteins into the telomeric interactome of A. thaliana.