Structural and Functional Characterization of the Conserved Nup82 Subcomplex Located on the Cytoplasmic Side of the Yeast Nuclear Pore Complex

Nuclear pore complexes (NPCs) are huge, proteinaceous assemblies, embedded in the nuclear envelope that act as gateways between the cytoplasm and nucleoplasm. A single NPC is formed from ~30 different nucleoporins, present in multiple copies. The vast majority of nucleoporins is organized in distinct subcomplexes. A number of NPC modules have been described in the past, but the structure of the tetrameric Nup82-Nup159C-Nsp1C-Dyn2 complex, located at the cytoplasmic side of the NPC and involved in nuclear mRNA export, remained elusive. My PhD study has focused on the conserved C-domain of Nup159, the key subunit of the Nup82 complex. Nup159-C harbors an essential -helical subdomain (H1), which is able to homo-dimerize through a coiled-coil interaction. Mutation in this region disturbs homo-dimerization and Nup82 complex assembly. The linker sequence prior to the essential H1 sub-domain turned out to be critical for dynein light chain (Dyn2) recruitment at the DIDNup159 motif (dynein light chain interacting domain), which binds five Dyn2 dimers. This finding suggests that DIDNup159 and H1 domains require a certain distance for Dyn2 binding to ensure the efficient Nup82 complex assembly and incorporation into the NPC scaffold. To gain more insight into the structure of the Nup82 complex, I have applied a method to reconstitute in vivo and subsequently isolate the essential structural core of the Nup82 module devoid of flexible FG repeats. Biochemical, biophysical and electron microscopy analysis unraveled that the Nup82 complex consists of a 20 nm stalk formed by the DIDNup159-Dyn2 and an asymmetric ’head’ structure that contains four Nup82 molecules with different properties and the helical C-domains of Nup159 and Nsp1, leading to a large assembly with a molecular mass in the megadalton range. Furthermore, cross-linking mass spectrometry allowed to map the interaction regions between four subunits of the complex. In conclusion, the study revealed for the first time the three dimensional (3D) architecture of the Nup82-Nup159C-Nsp1C-Dyn2 complex with Nup159 C-domain forming the key self-dimerizing scaffold

Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery

Elongator mutation in mice induces neurodegeneration and ataxia-like behavior

Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of diverse neurological disorders.The authors acknowledge the facilities, and the scientific and technical assistance of the Australian Phenomics Facility (APF), the Australian National University. The APF is supported by the Australian Phenomics Network (APN). The APN is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. We are very grateful to Jelena Bezbradica Mirkovic and Kate Schroder for providing NLRP3 KO and Caspase-1 KO animals and for their valuable discussion. We also thank Avril Robertson and Matthew Cooper for the gift of MCC950 and Trent Woodruff for advice regarding the administration of MCC950. We acknowledge Ting-Yu Lin and Andrzej Chramiec-Głąbik for providing labeled tRNAs. This work was supported by the POLONEZ1 Grant UMO-2015/19/P/NZ1/02514 from the National Science Centre, Poland and received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 665778 (M.G. and A.S.-K.) and the First Team grant First TEAM/ 2016-1/2 from the Foundation for Polish Science (S.G.)

Cryo-EM structure of the fully assembled elongator complex

Transfer RNA (tRNA) molecules are essential to decode messenger RNA codons during protein synthesis. All known tRNAs are heavily modified at multiple positions through post-transcriptional addition of chemical groups. Modifications in the tRNA anticodons are directly influencing ribosome decoding and dynamics during translation elongation and are crucial for maintaining proteome integrity. In eukaryotes, wobble uridines are modified by Elongator, a large and highly conserved macromolecular complex. Elongator consists of two subcomplexes, namely Elp123 containing the enzymatically active Elp3 subunit and the associated Elp456 hetero-hexamer. The structure of the fully assembled complex and the function of the Elp456 subcomplex have remained elusive. Here, we show the cryo-electron microscopy structure of yeast Elongator at an overall resolution of 4.3 Å. We validate the obtained structure by complementary mutational analyses in vitro and in vivo. In addition, we determined various structures of the murine Elongator complex, including the fully assembled mouse Elongator complex at 5.9 Å resolution. Our results confirm the structural conservation of Elongator and its intermediates among eukaryotes. Furthermore, we complement our analyses with the biochemical characterization of the assembled human Elongator. Our results provide the molecular basis for the assembly of Elongator and its tRNA modification activity in eukaryotes

Molecular insights into RNA recognition and gene regulation by the TRIM-NHL protein Mei-P26

The TRIM-NHL protein Meiotic P26 (Mei-P26) acts as a regulator of cell fate in Drosophila. Its activity is critical for ovarian germline stem cell maintenance, differentiation of oocytes, and spermatogenesis. Mei-P26 functions as a post-transcriptional regulator of gene expression; however, the molecular details of how its NHL domain selectively recognizes and regulates its mRNA targets have remained elusive. Here, we present the crystal structure of the Mei-P26 NHL domain at 1.6 Å resolution and identify key amino acids that confer substrate specificity and distinguish Mei-P26 from closely related TRIM-NHL proteins. Furthermore, we identify mRNA targets of Mei-P26 in cultured Drosophila cells and show that Mei-P26 can act as either a repressor or activator of gene expression on different RNA targets. Our work reveals the molecular basis of RNA recognition by Mei-P26 and the fundamental functional differences between otherwise very similar TRIM-NHL proteins

Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

Nuclear pore complexes (NPCs) are huge assemblies formed from ~30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery

Structural basis for assembly and function of the Nup82 complex in the nuclear pore scaffold

Nuclear pore complexes (NPCs) are huge assemblies formed from ∼30 different nucleoporins, typically organized in subcomplexes. One module, the conserved Nup82 complex at the cytoplasmic face of NPCs, is crucial to terminate mRNA export. To gain insight into the structure, assembly, and function of the cytoplasmic pore filaments, we reconstituted in yeast the Nup82–Nup159–Nsp1–Dyn2 complex, which was suitable for biochemical, biophysical, and electron microscopy analyses. Our integrative approach revealed that the yeast Nup82 complex forms an unusual asymmetric structure with a dimeric array of subunits. Based on all these data, we developed a three-dimensional structural model of the Nup82 complex that depicts how this module might be anchored to the NPC scaffold and concomitantly can interact with the soluble nucleocytoplasmic transport machinery

La polisemia y la sinonimia en el lenguaje jurídico. Estudio comparativo español-polaco

Casi todos los idiomas se caracterizan por la presencia de la sinonimia y la polisem ia que, a parte de ser una muestra de la riqueza lingüística del lenguaje en general, son las figuras muy vinculadas con los lenguajes de especialidad. El lenguaje jurídico que nos ocupará en el pres ente estudio, lejos de ser un í voco como muy a menudo se le atribuye, se caracteriza por la elevada presencia de ambos fenómenos. El objeto principal del nuestro trabajo es dar una prueba a esta afirmación a través de un análisis de los términos propios tan to para el lenguaje general como para el lenguaje jurídicoAlmost every language sufers from a presence of a s ynonymy and polysemy which are, besides of being a sign o f the lexical richness of language in general, are also the linguistic figures linked to specialized languages. The legal language that will interest us in this paper stays far away from transmitting the univocity that is usually attributed to it and it is characterized by more than a representative presence of both phenomena. In the present study we aim to prove this statement through the analysis of some terms proper for both general and legal language

LA POLISEMIA Y LA SINONIMIA EN EL LENGUAJE JURÍDICO – ESTUDIO COMPARATIVO ESPAÑOL-POLACO

Casi todos los idiomas se caracterizan por la presencia de la sinonimia y la polisemia que, aparte de ser una muestra de la riqueza lingüística del lenguaje en general, son las figuras muy vinculadas con los lenguajes de especialidad. El lenguaje jurídico que nos ocupará en el presente estudio, lejos de ser unívoco como muy a menudo se le atribuye, se caracteriza por la elevada presencia de ambos fenómenos. El objeto principal del nuestro trabajo es dar una prueba a esta afirmación a través de un análisis de los términos propios tanto para el lenguaje general como para el lenguaje jurídico

Marital status registration

Niniejsza praca została poświęcona aktom stanu cywilnego oraz kierownikowi urzędu stanu cywilnego jako osobie kompetentnej do sporządzania aktu urodzenia, małżeństwa i zgonu. Akty stanu cywilnego to pojedyncze wpisy w księgach stanu cywilnego, które rejestrują stan cywilny osoby poprzez odnotowanie pewnych określonych zdarzeń. Akty te stanowią wyłączny dowód zdarzeń w nich stwierdzonych, a ich niezgodność z prawdą może być udowodniona jedynie w postępowaniu sądowym. Sporządzenia każdego z aktów stanu cywilnego, ich odpisów oraz wzmianek dodatkowych w aktach stanu cywilnego dokonuje kierownik urzędu stanu cywilnego właściwy według miejsca zarejestrowanego zdarzenia, którym z mocy prawa jest każdy wójt, burmistrz albo prezydent miasta. Ze względu na szeroki zakres uprawnień oraz konieczność codziennej obecności w siedzibie urzędu stanu cywilnego, wójtowie, burmistrzowie oraz prezydenci miast powołują na to stanowisko inną osobę. Wysoka ranga tego stanowiska wymaga, aby kierownikiem urzędu stanu cywilnego została osoba która: posiada obywatelstwo polskie, jest nieposzlakowanej opinii, nie była prawomocnie skazana za przestępstwo umyślne, jej stan zdrowia pozwala na pełnienie tego urzędu oraz posiada niezbędne wykształcenie oraz doświadczenie w tym kierunku. Kierownik urzędu stanu cywilnego dokonuje wszystkich wpisów do aktów stanu cywilnego oraz zajmuje się rejestracją stanu cywilnego. Rejestracja ta stanowi element ewidencji ludności, która jest systemem obligatoryjnych, trwałych oraz ciągłych wpisów dotyczących stanu cywilnego. Rejestracji stanu cywilnego dokonuje się w księgach stanu cywilnego, które przechowywane są w siedzibie urzędu stanu cywilnego. Polskim aktem prawnym normującym problematykę aktów stanu cywilnego jest w chwili obecnej ustawa z dnia 29 września 1986 r. – prawo o aktach stanu cywilnego.The present work is devoted to the legal documents concerning marital status as well as the registrar as the person qualified to draw up a marriage or death certificate. The documents of marital status registration are single records in the registration books which register the marital status of a person on the basis of certain events. These records are the only proof of the events stated, therefore the untruthfulness of them can only be proved by a judicial verdict. The person capable of drawing up any of the legal documents concerning marital status or the copy of one, is the registrar of the place where the event registered occurred. By law, the registrar is any chief officer of a group of villages, mayor or town president. As it is obligatory for a registrar to stay at the office daily to fulfill a wide range of duties, the chief officers, mayors or town presidents usually appoint another person to than position. For the high rank of that position, it is obligatory that a person holding it fulfills certain conditions. They have to be of Polish citizenship and of impeccable reputation, they have to never had been finally sentenced for an intentional crime, their health condition has to enable them for the post, as well as they have to have the necessary education and experience in the field of dealing with marital status legal matters. The registrar deals with putting all the records of the marital status into the books of records, they also register the marital status of the citizens. The books above mentioned are the elements of population register, which is a system of obligatory, permanent or continuous records concerning the marital status of the population. All the data relating to marital status is recorded in the books of records and stored in the registry office. The legal act which currently regulates the issue of civil status documents is the act of law dated September, 29 th 1986 called the Law of Civil Status Acts