Final Report Summary - TRYPNUP (Nuclear envelope proteins of Trypanosoma brucei) In our project, we focused on the characterization of a nuclear lamina and other proteins that constitute the nuclear envelope in the unicellular parasite Trypanosoma brucei. The nuclear envelope (NE) is the defining feature of the eukaryotic cell and one of the major innovations differentiating eukaryotes from other life forms. The NE serves as a physical barrier separating the nuclear matrix from the cytoplasm and also functions in cell cycle progression and regulation of gene expression via interactions with the chromatin. A necessity for selective interchange of macro-molecules between the nucleoplasm and cytoplasm led to the evolution of transporting mechanisms consisting of ~30 protein subunits that form the nuclear pore complex (NPC). The NE is usually structurally supported with a meshwork of coiled-coil proteins that are called lamins in animals and are involved in the epigenetic regulation of gene expression. These nucleoskeletal proteins are connected with the cytoskeleton through SUN and KASH domain containing proteins that penetrate the inner and outer nuclear membrane, respectively. Until recently, all these structures were only known from organisms that belong to Opisthokonts, the eukaryotic supergroup that includes fungi and animals.Trypanosomes are members of the Excavata supergroup and likely diverged early in evolution after the last eukaryotic common ancestor (LECA). They constitute a significant proportion of global disease burden as causative agents of the African sleeping sickness and Chagas disease. They also have rather unusual mechanisms for controlling gene expression. The protein-coding genes are positioned in directional polycistonic transcription units (PTUs) and the resolution of individual mRNAs from PTU transcripts occurs by trans-splicing and subsequent polyadenylation. It was recently found that the NUP-1 protein, the major component of the trypanosomes’ NE, plays roles similar to metazoan lamins and is involved in the epigenetic regulation of developmentally regulated genes. Also the nuclear pore complex proteins of animals and fungi, especially their subset called nuclear basket, were previously shown to interact with a chromatin and promote gene expression and are also involved in regulating the mRNA export via interactions with other protein complexes. To better understand the evolution of these complex cellular structures and the protein interactions at the inner side of the trypanosome nuclear envelope, we investigated in more detail the functions of the NUP-1 protein, its interacting partner NUP-2 as well as the components of the trypanosome nuclear basket. From the phylogenetic distribution of NPC proteins (nucleoporins or Nups) we find that an NPC very similar to that in humans was already present in LECA. Very few Nups are lineage specific and some of the proteins that fail to be identified by in silico searches in diverged eukaryotes may be present as exemplified by proteomic studies on Trypanosoma, where many of the Nups with typical secondary structures were found despite the lack of sequence conservation. By contrast, several components likely evolved after the diversification of eukaryotes. The trypanosome nuclear basket, a dynamic subcomplex of the NPC facing the inside of the nucleus is composed of distinct proteins compared with animals and fungi. However, the functions of the basket are very similar and may thus serve as an example of convergent evolution. Further, this may also be the case for the NUP-1 protein, a trypanosome specific protein with roles similar to lamins, although architecture and sequence suggest no relationship. We found that like in lamins, NUP-1 phosphorylation status determines its state of polymerization and is important for the nuclear division during the cell cycle. NUP-2 protein, which interacts with NUP-1 is also essential for the growth of the parasite and its depletion by RNAi causes similar phenotype as for NUP-1. The nuclear envelope is disintegrated in similar way and the arrangement of the nuclear pore complexes is also affected. Moreover, the depletion of NUP-2 caused de-repression of the developmentally regulated genes such as the VSG coat proteins that are required by the parasite to escape from the recognition of the immune system of the host, which suggest that NUP-2 plays a role in epigenetic regulation in very similar way as NUP-1. The divergence of trypanosomes is further underlined by the apparent absence of the classical SUN/KASH domain protein complexes, which in other eukaryotes form bridge across the nuclear membranes and connect nuclear lamina with cytoskeleton. These proteins are otherwise conserved among eukaryotes and were clearly present in LECA. It is therefore apparent that the trypanosome nucleus functions using rather novel protein components in parallel with highly conserved elements. These findings have major implications for how the trypanosomatid nucleus operates and the evolution of the nucleus. The novel components, which are unique to the nucleus of trypanosomes and play roles essential for the survival of these parasites, might also provide much-needed targets for new anti-trypanosomal drugs.