The TELOMERE REPEAT BINDING proteins TRB4 and TRB5 function as transcriptional activators of PRC2-controlled genes to regulate plant development.

Plant-specific transcriptional regulators called TELOMERE REPEAT BINDING proteins (TRBs) combine two DNA-binding domains, the GH1 domain, which binds to linker DNA and is shared with H1 histones, and the Myb/SANT domain, which specifically recognizes the telobox DNA-binding site motif. TRB1, TRB2, and TRB3 proteins recruit Polycomb group complex 2 (PRC2) to deposit H3K27me3 and JMJ14 to remove H3K4me3 at gene promoters containing telobox motifs to repress transcription. Here, we demonstrate that TRB4 and TRB5, two related paralogs belonging to a separate TRB clade conserved in spermatophytes, regulate the transcription of several hundred genes involved in developmental responses to environmental cues. TRB4 binds to several thousand sites in the genome, mainly at transcription start sites and promoter regions of transcriptionally active and H3K4me3-marked genes, but, unlike TRB1, it is not enriched at H3K27me3-marked gene bodies. However, TRB4 can physically interact with the catalytic components of PRC2, SWINGER, and CURLY LEAF (CLF). Unexpectedly, we show that TRB4 and TRB5 are required for distinctive phenotypic traits observed in clf mutant plants and thus function as transcriptional activators of several hundred CLF-controlled genes, including key flowering genes. We further demonstrate that TRB4 shares multiple target genes with TRB1 and physically and genetically interacts with members of both TRB clades. Collectively, these results reveal that TRB proteins engage in both positive and negative interactions with other members of the family to regulate plant development through both PRC2-dependent and -independent mechanisms.

Evolutionarily conserved protein motifs drive interactions between the plant nucleoskeleton and nuclear pores.

The nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear shape and gene expression. To understand how the Arabidopsis (Arabidopsis thaliana) nucleoskeleton physically connects to the nuclear periphery in plants, we investigated the Arabidopsis nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. We identified 3 conserved peptide motifs within the N-terminal region of KAKU4, which are required for intermolecular interactions of KAKU4 with itself, interaction with the nucleoskeleton protein CROWDED NUCLEI (CRWN), localization at the nuclear periphery, and nuclear elongation in differentiated tissues. Unexpectedly, we find these motifs to be present also in NUP82 and NUP136, 2 plant-specific nucleoporins from the nuclear pore basket. We further show that NUP82, NUP136, and KAKU4 have a common evolutionary history predating nonvascular land plants with KAKU4 mainly localizing outside the nuclear pore suggesting its divergence from an ancient nucleoporin into a new nucleoskeleton component. Finally, we demonstrate that both NUP82 and NUP136, through their shared N-terminal motifs, interact with CRWN and KAKU4 proteins revealing the existence of a physical continuum between the nuclear pore and the nucleoskeleton in plants.

In Depth Topological Analysis of Arabidopsis Mid-SUN Proteins and Their Interaction with the Membrane-Bound Transcription Factor MaMYB.

Mid-SUN proteins are a neglected family of conserved type III membrane proteins of ancient origin with representatives in plants, animals, and fungi. Previous higher plant studies have associated them with functions at the nuclear envelope and the endoplasmic reticulum (ER). In this study, high-resolution confocal light microscopy is used to explore the localisation of SUN3 and SUN4 in the perinuclear region, to explore topology, and to study the role of mid-SUNs on endoplasmic reticulum morphology. The role of SUN3 in the ER is reinforced by the identification of a protein interaction between SUN3 and the ER membrane-bound transcription factor maMYB. The results highlight the importance of mid-SUNs as functional components of the ER and outer nuclear membrane.

Automated 3D bio-imaging analysis of nuclear organization by NucleusJ 2.0.

NucleusJ 1.0, an ImageJ plugin, is a useful tool to analyze nuclear morphology and chromatin organization in plant and animal cells. NucleusJ 2.0 is a new release of NucleusJ, in which image processing is achieved more quickly using a command-lineuser interface. Starting with large collection of 3D nuclei, segmentation can be performed by the previously developed Otsu-modified method or by a new 3D gift-wrapping method, taking better account of nuclear indentations and unstained nucleoli. These two complementary methods are compared for their accuracy by using three types of datasets available to the community at https://www.brookes.ac.uk/indepth/images/ . Finally, NucleusJ 2.0 was evaluated using original plant genetic material by assessing its efficiency on nuclei stained with DNA dyes or after 3D-DNA Fluorescence in situ hybridization. With these improvements, NucleusJ 2.0 permits the generation of large user-curated datasets that will be useful for software benchmarking or to train convolution neural networks.

Investigation of Nuclear Periphery Protein Interactions in Plants Using the Membrane Yeast Two-Hybrid (MbY2H) System.

Identification of membrane protein interactomes is a key issue to better understand how these molecules carry out their functions. However, protein-protein interactions using conventional interaction assays are particularly challenging for integral membrane proteins, because of their hydrophobic nature. Here we describe the membrane yeast two-hybrid (MbY2H) system, a powerful tool for identifying the interactors of membrane and membrane-associated proteins.

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its involvement in chromatin organisation has not been studied in plants. Here, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana in which heterochromatin clusters in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of the 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally, we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences.

A novel family of plant nuclear envelope-associated proteins.

This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

Characterization of two distinct subfamilies of SUN-domain proteins in Arabidopsis and their interactions with the novel KASH-domain protein AtTIK.

SUN-domain proteins belong to a gene family including classical Cter-SUN and mid-SUN subfamilies differentiated by the position of the SUN domain within the protein. Although present in animal and plant species, mid-SUN proteins have so far remained poorly described. Here, we used a combination of genetics, yeast two-hybrid and in planta transient expression methods to better characterize the SUN family in Arabidopsis thaliana. First, we validated the mid-SUN protein subfamily as a monophyletic group conserved from yeast to plant. Arabidopsis Cter-SUN (AtSUN1 and AtSUN2) and mid-SUN (AtSUN3 and AtSUN4) proteins expressed as fluorescent protein fusions are membrane-associated and localize to the nuclear envelope (NE) and endoplasmic reticulum. However, only the Cter-SUN subfamily is enriched at the NE. We investigated interactions in and between members of the two subfamilies and identified the coiled-coil domain as necessary for mediating interactions. The functional significance of the mid-SUN subfamily was further confirmed in mutant plants as essential for early seed development and involved in nuclear morphology. Finally, we demonstrated that both subfamilies interact with the KASH domain of AtWIP1 and identified a new root-specific KASH-domain protein, AtTIK. AtTIK localizes to the NE and affects nuclear morphology. Our study indicates that Arabidopsis Cter-SUN and mid-SUN proteins are involved in a complex protein network at the nuclear membranes, reminiscent of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex found in other kingdoms.

The plant LINC complex at the nuclear envelope.

Significant advances in understanding the plant nuclear envelope have been made over the past few years; indeed, knowledge of the protein network at the nuclear envelope is rapidly growing. One such network, the linker of nucleoskeleton and cytoskeleton (LINC) complex, is known in animals to connect chromatin to the cytoskeleton through the nuclear envelope. The LINC complex is made of Sad1/Unc84 (SUN) and Klarsicht/Anc1/Syne1 homology (KASH) proteins which have been recently characterized in plants. SUN proteins are located within the inner nuclear membrane, while the KASH proteins are included into the outer nuclear membrane. SUN and KASH domains interact and bridge the two nuclear membranes. In Arabidopsis, KASH proteins also interact with the tryptophan-proline-proline (WPP) domain-interacting tail-anchored protein 1 (WIT1), associated with the nuclear pore complex and with myosin XI-i which directly interacts with the actin cytoskeleton. Although evidence for a plant LINC complex connecting the nucleus to the cytoskeleton is growing, its interaction with chromatin is still unknown, but knowledge gained from animal models strongly suggests its existence in plants. Possible functions of the plant LINC complex in cell division, nuclear shape, and chromatin organization are discussed.

Identification and mapping of induced chromosomal deletions using sequence polymorphisms.

One of the many advantages of Drosophila melanogaster as a model organism is the relative ease with which gene deletions can be generated by imprecise excision of transposon insertions. Here, we describe a simple, fast, and efficient method of screening for single-gene excision events that is not biased by prior assumptions of the mutant phenotype. DNA sequence polymorphisms were used as co-dominant electrophoretic markers to identify candidate deletions in a single generation, and to delimit the breakpoints to within 0.5-1 kb, thereby rapidly identifying deficiencies that affect only the gene of interest. In addition, we used polymorphism profiling to map existing deficiencies. The method can also be applied to map the extent of deletions generated by x-rays and to identify targeted mutations generated by engineered zinc-finger nucleases in Drosophila and other polymorphic model organisms (e.g., zebrafish, mouse, Caenorhabditis elegans).

TOR regulates the subcellular distribution of DIM2, a KH domain protein required for cotranscriptional ribosome assembly and pre-40S ribosome export.

Eukaryotic ribosome synthesis is a highly dynamic process that involves the transient association of scores of trans-acting factors to nascent pre-ribosomes. Many ribosome synthesis factors are nucleocytoplasmic shuttling proteins that engage the assembly pathway at early nucleolar stages and escort pre-ribosomes to the nucleoplasm and/or the cytoplasm. Here, we report that two 40S ribosome synthesis factors, the KH-domain protein DIM2 and the HEAT-repeats/Armadillo-domain and export factor RRP12, are nucleolar restricted upon nutritional, osmotic, and oxidative stress. Nucleolar entrapment of DIM2 and RRP12 was triggered by rapamycin treatment and was under the strict control of the target of rapamycin (TOR) signaling cascade. DIM2 binds pre-rRNAs directly through its KH domain at the 5'-end of ITS1 (D-A(2) segment) and, consistent with its requirements in early nucleolar pre-rRNA processing, is required for efficient cotranscriptional ribosome assembly. The substitution of a single and highly conserved amino acid (G207A) within the KH motif is sufficient to inhibit pre-rRNA processing in a fashion similar to genetic depletion of DIM2. DIM2 carries an evolutionarily conserved putative nuclear export sequence (NES) at its carboxyl-terminal end that is required for efficient pre-40S ribosome export. Strikingly, DIM2 and RRP12 are both involved in the nucleocytoplasmic translocation of pre-ribosomes, suggesting that this step in the ribosome assembly pathway has been selected as a regulatory target for the TOR pathway.

Esf2p, a U3-associated factor required for small-subunit processome assembly and compaction.

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.

Dim2p, a KH-domain protein required for small ribosomal subunit synthesis.

Recent proteomic analyses are revealing the dynamics of preribosome assembly. Following cleavage at processing site A(2), which generates the 20S pre-rRNA (the immediate precursor to the 18S rRNA), early RRPs (ribosomal RNA processing factors) are released in bulk from the preribosomes, and the resulting pre-40S subunits are left associated with a limited set of proteins that we refer to as the SSU RRP complex. Dim2p, a core constituent of the SSU RRP complex and conserved KH-domain containing protein, is required for pre-rRNA processing and is associated with early nucleolar and late cytoplasmic pre-rRNA species. Consistently, Dim2p shuttles between the nucle(ol)us and the cytoplasm, a trafficking that is tightly regulated by growth. The association of Dim2p with the 18S rRNA dimethyltransferase Dim1p, as well as its requirement for pre-rRNA processing at cleavage sites A(1) and A(2) and for 18S rRNA dimethylation, suggest that Dim2p may recruit Dim1p to nucleolar pre-rRNAs through its KH domain.

Late cytoplasmic maturation of the small ribosomal subunit requires RIO proteins in Saccharomyces cerevisiae.

Numerous nonribosomal trans-acting factors involved in pre-rRNA processing have been characterized, but few of them are specifically required for the last cytoplasmic steps of 18S rRNA maturation. We have recently demonstrated that Rrp10p/Rio1p is such a factor. By BLAST analysis, we identified the product of a previously uncharacterized essential gene, YNL207W/RIO2, called Rio2p, that shares 43% sequence similarity with Rrp10p/Rio1p. Rio2p homologues were identified throughout the Archaea and metazoan species. We show that Rio2p is a cytoplasmic-nuclear protein and that its depletion blocks 18S rRNA production, leading to 20S pre-rRNA accumulation. In situ hybridization reveals that in Rio2p-depleted cells, 20S pre-rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. We also show that both Rio1p and Rio2p accumulate in the nucleus of crm1-1 cells at the nonpermissive temperature. Nuclear as well as cytoplasmic Rio2p and Rio1p cosediment with pre-40S particles. These results strongly suggest that Rio2p and Rrp10p/Rio1p are shuttling proteins which associate with pre-40S particles in the nucleus and they are not necessary for export of the pre-40S complexes but are absolutely required for the cytoplasmic maturation of 20S pre-rRNA at site D, leading to mature 40S ribosomal subunits.