Eukaryotic Ribosome assembly and Nucleocytoplasmic Transport

The process of eukaryotic ribosome assembly stretches across the nucleolus, the nucleoplasm and the cytoplasm, and therefore relies on efficient nucleocytoplasmic transport. In yeast, the import machinery delivers ~140,000 ribosomal proteins every minute to the nucleus for ribosome assembly. At the same time, the export machinery facilitates translocation of ~2000 pre-ribosomal particles every minute through ~200 nuclear pore complexes (NPC) into the cytoplasm. Eukaryotic ribosome assembly also requires >200 conserved assembly factors, which transiently associate with pre-ribosomal particles. Their site(s) of action on maturing pre-ribosomes are beginning to be elucidated. In this chapter, we outline protocols that enable rapid biochemical isolation of pre-ribosomal particles for single particle cryo-electron microscopy (cryo-EM) and in vitro reconstitution of nuclear transport processes. We discuss cell-biological and genetic approaches to investigate how the ribosome assembly and the nucleocytoplasmic transport machineries collaborate to produce functional ribosomes. Keywords: Budding Yeast; Nuclear Export; Nuclear Import; Ribosome Assembly; preribosome structure

Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2

Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2's interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channe

The ribotoxin α-sarcin can cleave the sarcin/ricin loop on late 60S pre-ribosomes

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.Spanish Ministry of Economy and Competitiveness MINECO and the European Union ERFD program [BFU2012-32404] to A.M.P., [BFU2016-75352-P

Targeted proteomics reveals compositional dynamics of 60S pre‐ribosomes after nuclear export

Construction and intracellular targeting of eukaryotic pre-ribosomal particles involve a multitude of diverse transiently associating trans-acting assembly factors, energy-consuming enzymes, and transport factors. The ability to rapidly and reliably measure co-enrichment of multiple factors with maturing pre-ribosomal particles presents a major biochemical bottleneck towards revealing their function and the precise contribution of >50 energy-consuming steps that drive ribosome assembly. Here, we devised a workflow that combines genetic trapping, affinity-capture, and selected reaction monitoring mass spectrometry (SRM-MS), to overcome this deficiency. We exploited this approach to interrogate the dynamic proteome of pre-60S particles after nuclear export. We uncovered assembly factors that travel with pre-60S particles to the cytoplasm, where they are released before initiating translation. Notably, we identified a novel shuttling factor that facilitates nuclear export of pre-60S particles. Capturing and quantitating protein interaction networks of trapped intermediates of macromolecular complexes by our workflow is a reliable discovery tool to unveil dynamic processes that contribute to their in vivo assembly and transport

Words cluster phonetically beyond phonotactic regularities

Recent evidence suggests that cognitive pressures associated with language acquisition and use could affect the organization of the lexicon. On one hand, consistent with noisy channel models of language (e.g., Levy, 2008), the phonological distance between wordforms should be maximized to avoid perceptual confusability (a pressure for dispersion). On the other hand, a lexicon with high phonological regularity would be simpler to learn, remember and produce (e.g., Monaghan et al., 2011) (a pressure for clumpiness). Here we investigate wordform similarity in the lexicon, using measures of word distance (e.g., phonological neighborhood density) to ask whether there is evidence for dispersion or clumpiness of wordforms in the lexicon. We develop a novel method to compare lexicons to phonotactically-controlled baselines that provide a null hypothesis for how clumpy or sparse wordforms would be as the result of only phonotactics. Results for four languages, Dutch, English, German and French, show that the space of monomorphemic wordforms is clumpier than what would be expected by the best chance model according to a wide variety of measures: minimal pairs, average Levenshtein distance and several network properties. This suggests a fundamental drive for regularity in the lexicon that conflicts with the pressure for words to be as phonologically distinct as possible. Keywords: Linguistics; Lexical design; Communication; Phonotactic

Nationalism in online sports journalism : A comparison between Germany and the UK

The focus of the study is an analysis, to what extend nationalist positionings are used in the online mediation of sports. The empirical study focuses on how nationality is represented and constructed in textual output. In addition, the study compares the findings of a content analysis conducted in Germany and the UK. As critical case the coverage of the 2012 UEFA EURO Championship qualification in 2011 has been selected.Previous research shows that nationalism in mediated sports within the traditional media is represented through various approaches. The usage of national identities and the construction of difference have been observed by researchers, such as Dixon (2000), Bruce (2004), Malcom (2009), Stocker (2009), Chesterton (2011) and others as mechanisms of nationalism. Furthermore, the developments and trends in the genre of sports journalism have been studied by researchers, such as Schlegel (2007) and Schmalenbach (2009), in regards to the journalistic profession, economical interests and tendencies in writing.The most important theory for this research has been the construction of difference in media output as well as how national identities or nationalism is represented in textual output. Most influential for this study are the theories by Stuart Hall (1997) and Teun van Dijk (1991). A definition regarding nationality and nationalism is based on the Danish researcher Jorgensen as presented by Östman (2009).The empirical study collects articles published on the two most visited websites / portals from Germany and the UK. 81 articles have been studied through a quantitative content analysis.From the empirical study it has been concluded that in both countries nationalist positionings are represented through constructions of difference. However, it needs to be noted that journalists in Germany and the UK are using different approaches. In Germany, country names and variations are used to create distance between the home and the other teams. In the UK they are used to strengthen their own national identity. Sources and quotations of the home teams are overrepresented to underline the connection between them and the audience. Pronouns and comparatives / superlatives are used to construct an “us-versus-them” relationship. Pronouns are more important in the UK for this approach, while in Germany it is mostly comparatives / superlatives. The frequencies of the different constructions vary, but all of them are used regularly in online sports reporting and confirm that there are nationalist tendencies in sports journalism