Telomerase Interaction Partners–Insight from Plants

Telomerase, an essential enzyme that maintains chromosome ends, is important for genome integrity and organism development. Various hypotheses have been proposed in human, ciliate and yeast systems to explain the coordination of telomerase holoenzyme assembly and the timing of telomerase performance at telomeres during DNA replication or repair. However, a general model is still unclear, especially pathways connecting telomerase with proposed non-telomeric functions. To strengthen our understanding of telomerase function during its intracellular life, we report on interactions of several groups of proteins with the Arabidopsis telomerase protein subunit (AtTERT) and/or a component of telomerase holoenzyme, POT1a protein. Among these are the nucleosome assembly proteins (NAP) and the minichromosome maintenance (MCM) system, which reveal new insights into the telomerase interaction network with links to telomere chromatin assembly and replication. A targeted investigation of 176 candidate proteins demonstrated numerous interactions with nucleolar, transport and ribosomal proteins, as well as molecular chaperones, shedding light on interactions during telomerase biogenesis. We further identified protein domains responsible for binding and analyzed the subcellular localization of these interactions. Moreover, additional interaction networks of NAP proteins and the DOMINO1 protein were identified. Our data support an image of functional telomerase contacts with multiprotein complexes including chromatin remodeling and cell differentiation pathways.

Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales, Cyanobacteria): a seaweed symbiont

This work introduces Waterburya agarophytonicola Bonthond and Shalygin gen. nov., sp. nov, a baeocyte producing cyanobacterium that was isolated from the rhodophyte Agarophyton vermiculophyllum (Ohmi) Gurgel et al., an invasive seaweed that has spread across the northern hemisphere. The new species genome reveals a diverse repertoire of chemotaxis and adhesion related genes, including genes coding for type IV pili assembly proteins and a high number of genes coding for filamentous hemagglutinin family (FHA) proteins. Among a genetic basis for the synthesis of siderophores, carotenoids and numerous vitamins, W. agarophytonicola is potentially capable of producing cobalamin (vitamin B12), for which A. vermiculophyllum is an auxotroph. With a taxonomic description of the genus and species and a draft genome, this study provides as a basis for future research, to uncover the nature of this geographically independent association between seaweed and cyanobiont.

PCMD-1 bridges the centrioles and the PCM scaffold in C. elegans

Correct cell division relies on the formation of a bipolar spindle. In animal cells, microtubule nucleation at the spindle poles is facilitated by the pericentriolar material (PCM), which assembles around a pair of centrioles. Although centrioles are essential for PCM assembly, proteins that anchor the PCM to the centrioles are less known. Here we investigate the molecular function of PCMD-1 in bridging the PCM and the centrioles in Caenorhabditis elegans. We demonstrate that the centrosomal recruitment of PCMD-1 is dependent on the outer centriolar protein SAS-7. While the most C-terminal part of PCMD-1 is sufficient to target it to the centrosome, the coiled-coil domain promotes its accumulation by facilitating self-interaction. We reveal that PCMD-1 is interacting with the PCM scaffold protein SPD-5, the mitotic kinase PLK-1 and the centriolar protein SAS-4. Using an ectopic translocation assay, we show that PCMD-1 can selectively recruit downstream PCM scaffold components to an ectopic location in the cell, indicating that PCMD-1 is able to anchor the PCM scaffold proteins at the centrioles. Our work suggests that PCMD-1 is an essential functional bridge between the centrioles and the PCM.

Phylogeny of Nitrogenase Structural and Assembly Components Reveals New Insights into the Origin and Distribution of Nitrogen Fixation across Bacteria and Archaea

The phylogeny of nitrogenase has only been analyzed using the structural proteins NifHDK. As nifHDKENB has been established as the minimum number of genes necessary for in silico prediction of diazotrophy, we present an updated phylogeny of diazotrophs using both structural (NifHDK) and cofactor assembly proteins (NifENB). Annotated Nif sequences were obtained from InterPro from 963 culture-derived genomes. Nif sequences were aligned individually and concatenated to form one NifHDKENB sequence. Phylogenies obtained using PhyML, FastTree, RapidNJ, and ASTRAL from individuals and concatenated protein sequences were compared and analyzed. All six genes were found across the Actinobacteria, Aquificae, Bacteroidetes, Chlorobi, Chloroflexi, Cyanobacteria, Deferribacteres, Firmicutes, Fusobacteria, Nitrospira, Proteobacteria, PVC group, and Spirochaetes, as well as the Euryarchaeota. The phylogenies of individual Nif proteins were very similar to the overall NifHDKENB phylogeny, indicating the assembly proteins have evolved together. Our higher resolution database upheld the three cluster phylogeny, but revealed undocumented horizontal gene transfers across phyla. Only 48% of the 325 genera containing all six nif genes are currently supported by biochemical evidence of diazotrophy. In addition, this work provides reference for any inter-phyla comparison of Nif sequences and a quality database of Nif proteins that can be used for identifying new Nif sequences.

Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

Characterization of major chromatin assembly proteins in tetrahymena thermophile using proeomic and molecular evolutionary analyses

Histones H3/H4 are deposited onto DNA in a replication-dependent or independent fashion by the CAF1 and HIRA protein complexes. Despite the identification of these protein complexes, mechanistic details remain unclear. Recently, we showed that in T. thermophila histone chaperones Nrp1, Asf1 and the Impβ6 importin function together to transport newly synthesized H3/H4 from the cytoplasm to the nucleus. To characterize chromatin assembly proteins in T.thermophila, I used affinity purification combined with mass spectrometry to identify protein-protein interactions of Nrp1, Cac2 subunit of CAF1, HIRA and histone modifying Hat1-complex in T. thermophila. I found that the three-subunit T.thermophila CAF1 complex interacts with Casein Kinase 2 (CKII), possibly accounting for previously reported human CAF1phosphorylation. I also found that Hat2 subunit of HAT1 complex is also shared by CAF1 complex as its Cac3 subunit. This suggests that Hat2/Cac3 might exist in two separate pools of protein complexes. Remarkably, proteomic analysis of Hat2/Cac3 in turn revealed that it forms several complexes with other proteins including SIN3, RXT3, LIN9 and TESMIN, all of which have known roles in the regulation of gene expression. Finally, I asked how selective forces might have impacted on the function of proteins involved in H3/H4 transport. Focusing on NASP which possesses several TPR motifs, I showed that its protein-protein interactions are conserved in T. thermophila. Using molecular evolutionary methods I show that different TPRs in NASP evolve at different rates possibly accounting for the functional diversity observed among different family members.

PCMD-1 bridges the centrioles and the PCM scaffold in C. elegans

ABSTRACTCorrect cell division relies on the formation of a bipolar spindle. In animal cells, microtubule nucleation at the spindle poles is facilitated by the pericentriolar material (PCM), which assembles around a pair of centrioles. Although centrioles are essential for PCM assembly, proteins that anchor the PCM to the centrioles are less known. Here we investigate the molecular function of PCMD-1 in bridging the PCM and the centrioles in Caenorhabditis elegans.We demonstrate that centrosomal recruitment of PCMD-1 is dependent on the outer centriolar protein SAS-7. While the most C-terminal part of PCMD-1 is sufficient to target it to the centrosome, the coiled-coil domain promotes its accumulation by facilitating self-interaction. We reveal that PCMD-1 is bridging the centrioles and PCM scaffold through protein-protein interactions with the PCM scaffold protein SPD-5, the mitotic kinase PLK-1 and the centriolar protein SAS-4. Using an ectopic translocation assay, we show that PCMD-1 is able to selectively recruit downstream PCM scaffold components to an ectopic location in the cell, indicating that PCMD-1 is sufficient to anchor the PCM scaffold proteins to the centrioles. Our work suggests that PCMD-1 is an essential functional bridge between the centrioles and the PCM.

Building the lipid droplet assembly complex

In this issue, Choudhary et al. (2020. J. Cell Biol.https://doi.org/10.1083/jcb.201910177) address the nature of the ER subdomain from which lipid droplets emanate and how several assembly proteins interact. Their data indicate that seipin/Nem1 marks these sites and provide a detailed working model for assembling the protein complex.