A comprehensive interaction map of the human survival of motor neuron (SMN) complex

Assembly of the Sm-class of U-rich small nuclear ribonucleoprotein particles (U snRNPs) is a process facilitated by the macromolecular survival of motor neuron (SMN) complex. This entity promotes the binding of a set of factors, termed LSm/Sm proteins, onto snRNA to form the core structure of these particles. Nine factors, including the SMN protein, the product of the spinal muscular atrophy (SMA) disease gene, Gemins 2-8 and unrip have been identified as the major components of the SMN complex. So far, however, only little is known about the architecture of this complex and the contribution of individual components to its function. Here, we present a comprehensive interaction map of all core components of the SMN complex based upon in vivo and in vitro methods. Our studies reveal a modular composition of the SMN complex with the three proteins SMN, Gemin8, and Gemin7 in its center. Onto this central building block the other components are bound via multiple interactions. Furthermore, by employing a novel assay, we were able to reconstitute the SMN complex from individual components and confirm the interaction map. Interestingly, SMN protein carrying an SMA-causing mutation was severely impaired in formation of the SMN complex. Finally, we show that the peripheral component Gemin5 contributes an essential activity to the SMN complex, most likely the transfer of Sm proteins onto the U snRNA. Collectively, the data presented here provide a basis for the detailed mechanistic and structural analysis of the assembly machinery of U snRNPs

Hvordan sykepleier kan fremme livskvalitet hos pårørende til mennesker med schizofreni

Bakgrunn for valg av tema Gjennom praksis og jobb i psykiatrien, har vi begge vært i kontakt med pårørende til mennesker med schizofreni. Vi har observert fortvilelse, angst, usikkerhet og håpløshet blant de nærmeste rundt pasienten. Vi opplever at håpløsheten hos de pårørende ofte er relatert til oppfatningen om den dårlige prognosen ved schizofreni. Fra erfaring, opplever vi generelt at pårørende til personer med alvorlig psykisk lidelse, fremstår som en neglisjert og nedprioritert gruppe. Sykepleier har en juridisk og etisk funksjon knyttet til å ivareta de pårørende. Hensikt med oppgaven Hensikten med oppgaven er undersøke hvordan sykepleier kan fremme livskvalitet i møte med pårørende. Oppgaven skal gi oss økt kunnskap om pårørendes situasjon, opplevelser og behov. Mål etter endt utdanning er å ivareta både pasienten og deres pårørende på en omsorgsfull måte. Metode Oppgaven er utformet som en integrativ litteraturstudie basert på vitenskapelige forskningsartikler, hvor dataene syntetiseres til en ny forståelse. Resultater Den første fasen rundt diagnostisering av schizofreni hos pasienten, kan medføre sjokk, sorg, bekymringer og helseplager for pårørende. Samtidig kan diagnostiseringen oppleves befriende ved at en får et navn på lidelsen, tilgang til helsehjelp og behandling, og mindre frykt for symptomene. Generelt har pårørende mange negative opplevelser i møte med helsevesenet, og det er behov for at helsepersonell yter bedre støtte og gir informasjon til pårørende. ACT-team kan bidra til bedre samarbeid, bekreftelse og åpenhet med pårørende. I den permanente rollen som pårørende kan mestringsstrategier være å mobilisere egenstyrke og motvekt, oppsøke støtte og samtale i pårørendeforeninger, hos profesjonelle eller blant familie og venner, og det å ta pause fra situasjonen. Diskusjon Resultatene indikerer behov for bedre ivaretakelse av pårørende i form av omsorg, oppfølging, informasjon og veiledning. Sykepleier må ha gode holdninger, som fremmer mestring og livskvalitet. Samtidig bør ansvaret for å inkludere, informere og samarbeide med pårørende, være en delegert oppgave

Reassessment of Piwi Binding to the Genome and Piwi Impact on RNA Polymerase II Distribution

Drosophila Piwi was reported by Huang et al. (2013) to be guided by piRNAs to piRNA-complementary sites in the genome, which then recruits Heterochromatin Protein 1a and histone methyltransferase Su(Var)3-9 to the sites. Among additional findings, Huang et al. (2013) also reported Piwi binding sites in the genome and the reduction of RNA polymerase II in euchromatin but its increase in pericentric regions in piwi mutants. Marinov et al. (2015) disputed the validity of the Huang et al. bioinformatic pipeline that led to the last two claims. Here we report our independent reanalysis of the data using current bioinformatic methods. Our reanalysis agrees with Marinov et al. (2015) that Piwi’s genomic targets still remain to be identified, yet confirms the Huang et al. claim that Piwi influences RNA polymerase II distribution in the genome. This Response addresses the Marinov et al. (2015) Matters Arising, published concurrently in Developmental Cell

Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m3G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m3G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m3G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA

An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a ∼21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes

Is there a common water-activity limit for the three domains of life?

Archaea and Bacteria constitute a majority of life systems on Earth but have long been considered inferior to Eukarya in terms of solute tolerance. Whereas the most halophilic prokaryotes are known for an ability to multiply at saturated NaCl (water activity (a w) 0.755) some xerophilic fungi can germinate, usually at high-sugar concentrations, at values as low as 0.650-0.605 a w. Here, we present evidence that halophilic prokayotes can grow down to water activities of <0.755 for Halanaerobium lacusrosei (0.748), Halobacterium strain 004.1 (0.728), Halobacterium sp. NRC-1 and Halococcus morrhuae (0.717), Haloquadratum walsbyi (0.709), Halococcus salifodinae (0.693), Halobacterium noricense (0.687), Natrinema pallidum (0.681) and haloarchaeal strains GN-2 and GN-5 (0.635 a w). Furthermore, extrapolation of growth curves (prone to giving conservative estimates) indicated theoretical minima down to 0.611 a w for extreme, obligately halophilic Archaea and Bacteria. These were compared with minima for the most solute-tolerant Bacteria in high-sugar (or other non-saline) media (Mycobacterium spp., Tetragenococcus halophilus, Saccharibacter floricola, Staphylococcus aureus and so on) and eukaryotic microbes in saline (Wallemia spp., Basipetospora halophila, Dunaliella spp. and so on) and high-sugar substrates (for example, Xeromyces bisporus, Zygosaccharomyces rouxii, Aspergillus and Eurotium spp.). We also manipulated the balance of chaotropic and kosmotropic stressors for the extreme, xerophilic fungi Aspergillus penicilloides and X. bisporus and, via this approach, their established water-activity limits for mycelial growth (∼0.65) were reduced to 0.640. Furthermore, extrapolations indicated theoretical limits of 0.632 and 0.636 a w for A. penicilloides and X. bisporus, respectively. Collectively, these findings suggest that there is a common water-activity limit that is determined by physicochemical constraints for the three domains of life

Boisliveau Anne-Sylvie, Le Coran par lui-même. Vocabulaire et argumentation du discours coranique autoréférentiel. Leyde, Brill, 2014

Neuenkirchen Paul. Boisliveau Anne-Sylvie, Le Coran par lui-même. Vocabulaire et argumentation du discours coranique autoréférentiel. Leyde, Brill, 2014. In: Bulletin critique des annales islamologiques, n°29, 2014. pp. 34-36

La fin du monde dans le Coran : une étude comparative du discours eschatologique coranique

Eschatology, or the discourse on the End, is arguably the Qur’an’s predominant thematic, giving vivid descriptions of cosmic and earthly cataclysms that will take place during the last days, depicting the Judgment as a horrific day for the sinners or portraying the final Resurrection in highly evocative terms. Paradoxically, this central qur’anic discourse regarding the End has little been discussed by modern scholars and has even been undervalued or even all together dismissed by a number of studies. Yet, according to scholars such as Paul Casanova (d. 1926), Tor Andrae (d. 1947) or more recently Stephen Shoemaker, eschatology should be regarded as the oldest strata of the Qur’an. This primitive layer would have been altered by the editors of the final version of this corpus after Muhammad’s death, when the End had not come. The object of my dissertation is to bring this eschatological discourse back in the spotlight by studying it in its historical context (i.e. that of Late Antiquity) as well as in its literary context (i.e. that of religious writings from the “Biblical” tradition). By doing so, I wish on the one hand to study the qur’anic text in itself, without its later traditional Muslim interpretations which always read the Qur’an in light of a mythified life of Muhammad. On the other hand I seek to understand the Qur’an in the continuity of previous religious eschatological texts. Many Western scholars have long noticed the similarities between certain biblical narratives (from the ‘canonical’ books of the Old and New Testaments) and the Qur’an, at the same time insisting on discrepancies between the two versions. These differences have often been said to be the result of Muhammad’s or someone else’s mistake. With the present study, I wish to show that the Qur’an is not “influenced” by biblical narratives as has often been thought. Rather, I believe that its authors have composed a novel writing in rhymed and rhythmic speech, based on one or more biblical subtext(s), very much like what Christian homilists did. As Gabriel Said Reynolds has argued, the fundamental medium of exhortation in their homilies is the use of eschatology. I therefore suggest to read the numerous verses of the Qur’an that deal with the End in light of a small corpus of eschatological homilies written a little more than a century before the Qur’an by Narsai (d. ca. 502) and Jacob of Serugh (d. 521), two Christian authors who wrote in Syriac. It is my hope to shed a light both on shared rhetorical techniques used by these authors and those of the Qur’an as well as on some ambiguous or problematic aspects of the qur’anic eschatological discourse.L’eschatologie, ou le discours sur la Fin, est sans nul doute la thématique centrale du Coran, allant des descriptions saisissantes des bouleversements cosmiques et terrestres qui auront lieu aux jours derniers, aux scènes du Jugement terrible pour les impies, en passant par les scènes imagées de la Résurrection. Paradoxalement, cet important discours coranique sur la Fin a été très peu abordé par la recherche universitaire, voir même occulté par certains travaux. Pourtant, de l’avis de certains chercheurs tels que Paul Casanova (m. 1926), Tor Andrae (m. 1947) ou plus récemment, Stephen Shoemaker, l’eschatologie constituerait la partie primitive du Coran, une partie qui aurait été altérée par les éditeurs de la version finale du corpus à la mort de Muhammad lorsque la Fin n’était pas venue. L’objet de ma thèse est de remettre ce discours eschatologique au premier plan en le replaçant dans son contexte historique (celui de l’Antiquité Tardive) et littéraire (celui des écrits religieux de tradition « biblique »). Ce faisant je souhaite d’une part amener un regard sur le texte coranique qui ne repose pas entièrement sur les sources traditionnelles musulmanes postérieures qui le lisent à la lumière d’évènements liés à la vie de Muhammad qui sont le plus souvent mythifiés, et d’autre part je cherche à l’inscrire dans la continuité des textes religieux antérieurs qui décrivent la Fin du monde. Depuis longtemps, nombreux sont les chercheurs occidentaux à avoir noté les similitudes entre certains récits bibliques (tirés des livres ‘canoniques’ de l’Ancien et du Nouveau Testaments) et le Coran, remarquant dans le même temps la présence d’écarts considérables entre les deux versions. Ces disparités ont souvent été mises sur le compte d’erreurs de compréhension due à Muhammad ou à son entourage. Dans le cadre de cette thèse, je voudrais suggérer que le texte du Coran n’est pas « influencé » par ces écrits bibliques comme cela a souvent été avancé, mais plutôt qu’à la manière des homélistes chrétiens, ses auteurs ont composé un texte original rimé et rythmé en prenant comme sous-texte un ou plusieurs verset(s) biblique(s). Comme Gabriel Said Reynolds l’a constaté, l’homéliste livre essentiellement son exhortation par le biais du discours eschatologique. Ainsi, je propose de lire les nombreux versets coraniques traitant de la Fin à la lumière d’un petit corpus d’homélies eschatologiques composées un peu plus d’un siècle avant le Coran par Narsaï (m. ca. 502) et Jacques de Saroug (m. 521), deux auteurs chrétiens de langue syriaque. Ce faisant je voudrais apporter un éclairage à la fois sur les techniques rhétoriques communes employées par ces auteurs et ceux du Coran, ainsi que sur de nombreuses zones d’ombre liées au vocabulaire et aux images employées dans le discours eschatologique coranique

The end of the world in the Quran : a comparative study of the quranic eschatological discourse

L’eschatologie, ou le discours sur la Fin, est sans nul doute la thématique centrale du Coran, allant des descriptions saisissantes des bouleversements cosmiques et terrestres qui auront lieu aux jours derniers, aux scènes du Jugement terrible pour les impies, en passant par les scènes imagées de la Résurrection. Paradoxalement, cet important discours coranique sur la Fin a été très peu abordé par la recherche universitaire, voir même occulté par certains travaux. Pourtant, de l’avis de certains chercheurs tels que Paul Casanova (m. 1926), Tor Andrae (m. 1947) ou plus récemment, Stephen Shoemaker, l’eschatologie constituerait la partie primitive du Coran, une partie qui aurait été altérée par les éditeurs de la version finale du corpus à la mort de Muhammad lorsque la Fin n’était pas venue. L’objet de ma thèse est de remettre ce discours eschatologique au premier plan en le replaçant dans son contexte historique (celui de l’Antiquité Tardive) et littéraire (celui des écrits religieux de tradition « biblique »). Ce faisant je souhaite d’une part amener un regard sur le texte coranique qui ne repose pas entièrement sur les sources traditionnelles musulmanes postérieures qui le lisent à la lumière d’évènements liés à la vie de Muhammad qui sont le plus souvent mythifiés, et d’autre part je cherche à l’inscrire dans la continuité des textes religieux antérieurs qui décrivent la Fin du monde. Depuis longtemps, nombreux sont les chercheurs occidentaux à avoir noté les similitudes entre certains récits bibliques (tirés des livres ‘canoniques’ de l’Ancien et du Nouveau Testaments) et le Coran, remarquant dans le même temps la présence d’écarts considérables entre les deux versions. Ces disparités ont souvent été mises sur le compte d’erreurs de compréhension due à Muhammad ou à son entourage. Dans le cadre de cette thèse, je voudrais suggérer que le texte du Coran n’est pas « influencé » par ces écrits bibliques comme cela a souvent été avancé, mais plutôt qu’à la manière des homélistes chrétiens, ses auteurs ont composé un texte original rimé et rythmé en prenant comme sous-texte un ou plusieurs verset(s) biblique(s). Comme Gabriel Said Reynolds l’a constaté, l’homéliste livre essentiellement son exhortation par le biais du discours eschatologique. Ainsi, je propose de lire les nombreux versets coraniques traitant de la Fin à la lumière d’un petit corpus d’homélies eschatologiques composées un peu plus d’un siècle avant le Coran par Narsaï (m. ca. 502) et Jacques de Saroug (m. 521), deux auteurs chrétiens de langue syriaque. Ce faisant je voudrais apporter un éclairage à la fois sur les techniques rhétoriques communes employées par ces auteurs et ceux du Coran, ainsi que sur de nombreuses zones d’ombre liées au vocabulaire et aux images employées dans le discours eschatologique coranique.Eschatology, or the discourse on the End, is arguably the Qur’an’s predominant thematic, giving vivid descriptions of cosmic and earthly cataclysms that will take place during the last days, depicting the Judgment as a horrific day for the sinners or portraying the final Resurrection in highly evocative terms. Paradoxically, this central qur’anic discourse regarding the End has little been discussed by modern scholars and has even been undervalued or even all together dismissed by a number of studies. Yet, according to scholars such as Paul Casanova (d. 1926), Tor Andrae (d. 1947) or more recently Stephen Shoemaker, eschatology should be regarded as the oldest strata of the Qur’an. This primitive layer would have been altered by the editors of the final version of this corpus after Muhammad’s death, when the End had not come. The object of my dissertation is to bring this eschatological discourse back in the spotlight by studying it in its historical context (i.e. that of Late Antiquity) as well as in its literary context (i.e. that of religious writings from the “Biblical” tradition). By doing so, I wish on the one hand to study the qur’anic text in itself, without its later traditional Muslim interpretations which always read the Qur’an in light of a mythified life of Muhammad. On the other hand I seek to understand the Qur’an in the continuity of previous religious eschatological texts. Many Western scholars have long noticed the similarities between certain biblical narratives (from the ‘canonical’ books of the Old and New Testaments) and the Qur’an, at the same time insisting on discrepancies between the two versions. These differences have often been said to be the result of Muhammad’s or someone else’s mistake. With the present study, I wish to show that the Qur’an is not “influenced” by biblical narratives as has often been thought. Rather, I believe that its authors have composed a novel writing in rhymed and rhythmic speech, based on one or more biblical subtext(s), very much like what Christian homilists did. As Gabriel Said Reynolds has argued, the fundamental medium of exhortation in their homilies is the use of eschatology. I therefore suggest to read the numerous verses of the Qur’an that deal with the End in light of a small corpus of eschatological homilies written a little more than a century before the Qur’an by Narsai (d. ca. 502) and Jacob of Serugh (d. 521), two Christian authors who wrote in Syriac. It is my hope to shed a light both on shared rhetorical techniques used by these authors and those of the Qur’an as well as on some ambiguous or problematic aspects of the qur’anic eschatological discourse

Die Biogenese kleiner nukleärer Ribonukleinprotein Partikel - ein in vitro System

Most protein-encoding genes in Eukaryotes are separated into alternating coding and non-coding sequences (exons and introns). Following the transcription of the DNA into pre-messenger RNA (pre-mRNA) in the nucleus, a macromolecular complex termed spliceosome removes the introns and joins the exons to generate mature mRNA that is exported to the cytoplasm. There, it can be interpreted by ribosomes to generate proteins. The spliceosome consists of five small nuclear ribonucleic acids (snRNAs) and more than 150 proteins. Integral components of this complex are RNA-protein particles (RNPs) composed of one or two snRNAs, seven common (Sm) and a various number of snRNP-specific proteins. The Sm proteins form a ring-structure around a conserved site of the snRNA called Sm site. In vitro, Sm proteins (B/B', D1, D2, D3, E, F, G) and snRNA readily assemble to form snRNPs. In the context of the cell, however, two macromolecular trans-acting factors, the PRMT5 (protein arginine methyltransferases type 5) and the SMN (survival motor neuron) complex, are needed to enable this process. Initially, the Sm proteins in the form of heterooligomers D1/D2, D3/B and F/E/G are sequestered by the type II methyltransferase PRMT5. pICln, a component of the PRMT5 complex, readily interacts with Sm proteins to form two distinct complexes. Whereas the first one comprises pICln and D3/B the second one forms a ring consisting of pICln, D1/D2 and F/E/G (6S). It has been found that pICln prevents the premature interaction of snRNAs with the Sm proteins in these complexes and thus functions as an assembly chaperone imposing a kinetic trap upon the further assembly of snRNPs. PRMT5 catalyzes the symmetrical dimethylation of arginine residues in B/B', D1 and D3 increasing their affinity towards the SMN complex. Finally, the SMN complex interacts with the pICln-Sm protein complexes, expels pICln and mediates snRNP assembly in an ATP-dependent reaction. So far, only little is known about the action of PRMT5 in the early phase of snRNP assembly and especially how the 6S complex is formed. Studies of this have so far been hampered by the unavailability of soluble and biologically active PRMT5 enzyme. The composition of the SMN complex and possible functions of individual subunits have been elucidated or hypothesized in recent years. Still, the exact mechanism of the entire machinery forming snRNPs is poorly understood. In vivo, reduced production of functional SMN protein results in the neurodegenerative disease spinal muscular atrophy (SMA). How specific SMN mutations that have been found in SMA patients cause the disease remains elusive, yet, are likely to interfere with either SMN complex stability or snRNP assembly. The aim of this work was to establish an in vitro system to recapitulate the cytoplasmic assembly of snRNPs. This was enabled by the recombinant production of all PRMT5 and SMN complex components as well as Sm proteins in a combination of bacterial and insect cell expression systems. Co-expression of human PRMT5 and its direct interaction partner WD45 (WD-repeat domain 45) in Sf21 (Spodoptera frugiperda 21) insect cells resulted for the first time in soluble and biologically active enzyme. Recombinant PRMT5/WD45 formed complexes with Sm protein heterooligomers as well as pICln-Sm protein complexes but not with F/E/G alone. Also, the enzyme exhibited a type II methyltransferase activity catalyzing the mono- (MMA) and symmetrical dimethylation (sDMA) of Sm proteins B, D1 and D3. Two experimental setups were devised to quantitatively analyze the overall methylation of substrates as well as to identify the type and relative abundance of specific methylation types. Methylation of Sm proteins followed Michaelis-Menten kinetics. Complex reconstitutions and competition of the methylation reaction indicate that 6S is formed in a step-wise manner on the PRMT5 complex. The analysis of the methylation type could be applied to deduce a model of sequential MMA and sDMA formation. It was found that large Sm protein substrate concentrations favored monomethylation. Following a distributive mechanism this leads to the conclusion that PRMT5 most likely confers partial methylation of several different substrate proteins instead of processing a single substrate iteratively until it is completely dimethylated. Finally, the human SMN complex was reconstituted from recombinant sources and was shown to be active in snRNP formation. The introduction of a modified SMN protein carrying a mutation (E134K) present in spinal muscular atrophy (SMA) proved that mutated complexes can be generated in vitro and that these might be applied to elucidate the molecular etiology of this devastating disease.Der Großteil der Protein-kodierenden Gene in Eukaryoten ist in kodierende und nicht-kodierende Regionen unterteilt - sogenannte Exons und Introns. Damit aus einem Gen ein Protein hergestellt werden kann, muss zunächst die genomische DNA im Rahmen der Translation in prä-messenger RNA (prä-mRNA; Boten-RNA) übersetzt werden. Aus dieser prä-mRNA werden anschließend durch einen makromolekularen Komplex (Spleißosom) die Introns entfernt und die kodieren Exons zusammengefügt. Die daraus resultierende gereifte mRNA dient letztendlich den Ribosomen als Vorlage zur Herstellung von Proteinen. Das Spleißosom besteht aus fünf snRNAs (small nuclear ribonucleic acids) und über 150 weiteren Proteinen. Zentrale Komponenten dieses Komplexes sind RNA-Protein Partikel (RNPs), die aus einer bzw. zwei snRNAs, sieben gemeinsamen (Sm) und weiteren snRNP-spezifischen Proteinen bestehen. Die Sm Proteine (B/B', D1, D2, D3, E, F and G) bilden eine Ringstruktur um eine konservierte Sequenz (Sm-site) der snRNA aus. In vitro erfolgt die Ausbildung dieser Struktur spontan. Im zellulären Kontext wird die Zusammenlagerung dieser snRNPs allerdings erst durch zwei makromolekulare, trans-agierende Proteinkomplexe, den PRMT5 und den SMN Komplex, ermöglicht. Zu Beginn interagieren die Sm Proteine als heterooligomere Strukturen bestehend aus D1/D2, D3/B und F/E/G mit der Typ II Methyltransferase PRMT5. pICln, eine Komponente des PRMT5 Komplexes, interagiert mit den Sm Proteinen und bildet zwei spezifische Komplexe aus. Während der erste aus pICln und D3/B besteht, lagern sich im zweiten die Sm proteine D1/D2 und F/E/G mit pICln zu einem Ring zusammen (6S Komplex). Diese Interaktion erzeugt eine kinetische Falle, so dass die Sm Proteine sich nicht mehr spontan an die snRNA anlagern können und somit die snRNP Biogenese verzögert wird. PRMT5 katalysiert die symmetrische Dimethylierung von Argininresten in B/B', D1 und D3, wodurch deren Affinität zum SMN Komplex erhöht wird. Letztendlich assoziert der SMN Komplex mit den zuvor erzeugten pICln-Sm Protein Komplexen, entlässt pICln und ermöglicht im weiteren die Zusammenlagerung von snRNPs in einer ATP-abhängigen Reaktion. Aktuell ist über die Funktion von PRMT5 in der frühen Phase der snRNP Biogenese wenig bekannt. Dies trifft insbesondere auf die Zusammenlagerung des 6S Komplexes zu. Biochemische Untersuchungen waren bis jetzt nahezu unmöglich, da rekombinant hergestelltes Protein entweder unlöslich oder biochemisch inaktiv war. In den vergangenen Jahren wurde viel über die Zusammensetzung des SMN Komplexes sowie über die Funktionen einzelner Untereinheiten herausgefunden aber auch spekuliert. Trotz alledem ist der genaue Mechanismus der snRNP Biogenese noch nahezu unbekannt. In vivo sind verringerte Mengen an funktionalem SMN Protein der Ausschlaggeber für die neurodegenerative Krankheit Spinale Muskelatrophie (SMA). Welchen Effekt Mutationen im SMN Protein haben, die in SMA Patienten festgestellt wurden ist ungewiss. Es ist allerdings zu vermuten, dass diese entweder die Integrität des SMN Komplexes negativ beeinflussen oder störend auf die snRNP Biogenese wirken. Das Ziel dieser Arbeit war es ein in vitro-System zu generieren, um die zytoplasmatische snRNP Biogenese biochemisch zu untersuchen. Dies geschah durch die rekombinante Produktion aller PRMT5 und SMN Komplex Komponenten sowie der Sm Proteine in einer Kombination von bakterieller und Insektenzell-Expression. Durch die Ko-Expression von humanem PRMT5 und dem Interaktionspartner WD45 (WD-repeat domain 45) in Sf21 (Spodoptera frugiperda 21) Insekten Zellen konnte erstmals lösliches und enzymatisch aktives Protein hergestellt werden. Rekombinantes PRMT5/WD45 bildete Komplexe mit heterooligomeren Sm Proteinen sowie pICln-Sm Protein Komplexen, allerdings nicht mit F/E/G. Zusätzlich konnte eine Typ II Methyltransferase Aktivität dadurch nachgewiesen werden, dass die Sm Protein B, D1 und D3 monomethyliert (MMA) und symmetrisch dimethyliert (sDMA) werden können. Zur weiteren Untersuchung wurden zwei experimentelle Ansätze erarbeitet, um die allgemeine Methylierungsaktivität sowie das relative Vorhandensein von Mono- und Dimethylargininen zu bestimmen. Es konnte gezeigt werden, dass die Methylierung der Sm Proteine einer Michael-Menten Kinetik folgt. Die Rekonstitution von PRMT-Sm Protein Komplexen sowie the Methylierungsreaktionen deuten auf eine schrittweise Zusammenlagerung von 6S auf dem PRMT5 Komplex hin. ..