A role for protein phosphatase PP1γ in SMN complex formation and subnuclear localization to Cajal bodies

The spinal muscular atrophy (SMA) gene product SMN forms with gem-associated protein 2-8 (Gemin2-8) and unrip (also known as STRAP) the ubiquitous survival motor neuron (SMN) complex, which is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and their localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs is reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using co-immunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1γ. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1γ to CBs. Moreover, depletion of PP1γ by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1γ-depleted cells. Two-dimensional protein gel electrophoresis revealed that SMN is hyperphosphorylated in nuclear extracts of PP1γ-depleted cells and expression of PP1γ restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1γ in the atrophic skeletal muscles, suggesting that the function of PP1γ is likely to be affected in disease. Our findings reveal a role of PP1γ in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1γ as a target for therapeutic intervention in SMA

GENERAZIONE E CORREZIONE DI CELLULE STAMINALIPLURIPOTENTI INDOTTE NON VIRALI DA PAZIENTE SMA COMEMODELLO DI MALATTIA E SORGENTE PER LA TERAPIA CELLULARE

Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases causing infant mortality. SMA is an autosomal recessive genetic disorder caused by mutations in the survival motor neuron 1 gene (SMN1), leading to the depletion of survival motor neuron (SMN) protein and resulting in the selective degeneration of spinal cord motor neurons. Patients with SMA exhibit muscle weakness and hypotonia. There is no cure for this disorder, which is devastating for patients and their families and a serious societal health problem. The human genome also harbors the SMN2 gene, which is almost identical to SMN1 except for a single nucleotide difference in SMN2. The splicing change resulting from this difference yields only 10% of the full-length protein and high levels of an unstable, truncated protein lacking exon 7 (SMNDelta7). Although worms, flies, and mice are useful for studying disease pathogenesis and drug screening, they have important limitations in recapitulating human diseases. One is that they lack SMN2, which can be introduced only with transgenic modifications. The possibility of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) has enabled derivation of disease-specific pluripotent cells, offering unprecedented access to modeling human disease and for cell and gene therapy applications. Here, we successfully generated human SMA-iPSCs from a type 1 SMA patient and his unaffected father, using non-integrating episomal vectors and demonstrated their differentiation into motoneurons. Moreover, we employed single-stranded oligonucleotides to correct defective SMN1, the SMA gene, using SMN2. Corrected cell lines contained no exogenous sequences and appeared indistinguishable from healthy iPSCs. Non-viral SMA-iPSC-derived motor neurons reproduced disease-specific features while corrected SMA-specific-iPSCs gave rise to phenotypically rescued motor neurons in vitro and in vivo. Our next goal was to determine whether MNs derived from iPSCs survive and engraft appropriately within the SMA spinal cord, the effect of disease environment on grafted cells and vice versa, and whether transplantation can ameliorate the disease phenotype in SMA transgenic mice. iPSC-purified motoneurons were used for transplantation into the spinal cords of 1-day-old SMA mice. Transplantation of wild-type and corrected SMA motor neurons extended lifespan and ameliorated the phenotype of SMA mice. These results offer proof-of-concept that generating patient-specific iPSCs and motor neurons free of exogenous elements may be possible, with potential for research and clinical applications

Structural Studies of the SMN-Gemin2 Complex

The proteins SMN and Gemin2 form the conserved core of the larger eponymous SMN complex, which also contains Gemins3-8 and unrip in an unknown stoichiometry. The complex facilitates the ordered assembly of seven Sm proteins onto a conserved site of an snRNA to form spliceosomal snRNP cores. A deficit in functional SMN as a consequence of deletions or loss-of-function mutations in the gene SMN1 underlies the disease spinal muscular atrophy (SMA), a severe neurodegenerative disorder and leading genetic cause of infant mortality. Here, the NMR solution structure of the interacting domains of human SMN and Gemin2 is reported. The Gemin2-SMN heterodimer forms a novel all α-helical fold, comprising a single SMN helix embedded in an elongated, hydrophobic cavity of Gemin2. The interface between SMN and Gemin2 is mediated through highly conserved residues on the hydrophobic face of the amphipathic SMN helix and on four Gemin2 helices far separated in primary sequence. Mutations of interfacial residues predicted by the structure to be essential, but not of known disease mutations, disrupted the SMN-Gemin2 interaction. A number of biophysical studies also showed that unbound Gemin2 undergoes a significant conformational change upon SMN binding to become more compact, but belied reports of a Gemin2 self-interaction. A related set of investigations conducted on the full-length orthologous S. pombe Gemin2-SMN proteins showed that the complex occupies small, discrete oligomers that self-associate to form larger assemblies; its apparently large molecular weight by analytical size-exclusion chromatography results from an elongated, partially unstructured state. Together, these NMR and biophysical studies of SMN and Gemin2 provide the first high-resolution glimpse of the SMN-Gemin2 heterodimer, establish a framework for future structure-function studies investigating snRNP biogenesis and SMA pathogenesis and provide new valuable insight into the overall oligomerization state of the SMN complex

Quantitative Analyse des Survival of Motor Neuron(SMN)-Komplexes unter Verwendung von absoluter Quantifizierung SMN-Komplex-spezifischer Peptide durch synthetische stabilisotopenmarkierte Peptidanaloga

Der SMN-Komplex ist eine makromolekulare Einheit, die aus neun festen Untereinheiten (SMN, Gemin2-Gemin8, Unrip) und verschiedenen transienten Faktoren (SmB/B’, SmE, SmF, SmG, SmD1-D3) aufgebaut ist. Die Hauptaufgabe des ubiquitären Komplexes umfasst die Assemblierung der U snRNPs im Cytoplasma und ihre Translokation in den Zellkern, wo sie am Aufbau des Spleißosoms beteiligt sind. Durch Mutation oder Deletion des SMN1-Gens kann dieser Prozess gestört sein und dadurch Spinale Muskelatrophie (SMA) auslösen. SMA ist die zweithäufigste autosomal rezessiv vererbbare Krankheit mit Todesfolge nach Mukoviszidose, die zu einer Degeneration der -Motorneuronen des Rückenmarks und damit zu einer Schwächung und einem Abbau der Muskulatur führt. Das Ziel der vorliegenden Arbeit war die Charakterisierung des humanen Survival of Motor Neuron-Komplex (SMN) hinsichtlich Zusammensetzung und Stöchiometrie mittels Anwendung von massenspektrometrischen Methoden. In diesem Zusammenhang wurde die native stöchiometrische Zusammensetzung des zentralen SMN-Komplexes mit zwei rekombinanten Komplexen verglichen, die beide eine in Verbindung mit SMA bekannte Mutation (Y272C, E134K) integriert hatten. Zu diesem Zweck wurden Wildtyp-SMN und die mutierten SMN-Analoga als GST-Fusions-Proteine zusammen mit Wildtyp-Gemin2, -Gemin6, -Gemin7 und -Gemin8 in Escherichia coli exprimiert. Nach Ernte und Lyse der Zellen wurde SMN auf einer Glutathion- Sepharose-Matrix angereichert und anschließend mittels verschiedener Methoden (Carbamidomethylierung, Trypsin-Verdau) für die massenspektrometrische quantitative Analyse vorbereitet. Des Weiteren wurden die Unterschiede zwischen dem cytoplasmatischen und dem nukleären SMN-Komplex, welche durch Zellkern-Cytoplasma-Trennung von HeLa-Zellen mittels eines modifizierten Roeder-Protokolls und anschließende Co-Immunopräzipitation gegen den SMNspezifischen Antikörper 7B10 erhalten wurden, untersucht. Für die Quantifizierung der SMN-Komplexe wurde Absolute Quantifizierung (AQUA) angewendet. Synthetische stabilisotopenmarkierte Petidanaloga wurden mit den nativen Proben in definierten Konzentrationen verdünnt und konnten während der massenspektrometrischen Analyse durch ihre Massendifferenz von 6 bis 10 Dalton im Vergleich zu den endogenen Peptiden detektiert werden. Zuvor mussten jedoch geeignete Peptidsequenzen ausgewählt werden, um eine reproduzierbare Quantifizierung zu gewährleisten. Dazu mussten die Peptide definierten Spezifikationen entsprechen (z.B. keine leicht modifizierbaren Aminosäuren oder keine überlesenen tryptischen Schnittstellen beinhalten). Die MS-Analyse wurde mittels Selected Reaction Monitoring (S/MRM) auf zwei unterschiedlichen Triple-Quadrupol-Systemen durchgeführt. Zu diesem Zweck wurden die Übergänge und Systemparameter (Declustering Potential und Kollisionsenergie) für jedes einzelne Peptid optimiert und festgelegt. Alternativ wurde markierungsfreie Quantifizierung auf einem LTQ Obitrap Velos System angewandt. Die hohe Massengenauigkeit und Sensitivität des Hybrid-FT-MS-Systems erlaubt eine Quantifizierung der entsprechenden Mutterionen direkt aus der MS1-Spur (ohne Anwendung von Tandem-MS-Experimenten). Beide Strategien, S/MRM und MS1, erlauben eine Quantifizierung von Peptiden mit Konzentrationen bis 250 amol. Durch Optimierung des dargestellten Protokolls, war es möglich den Einfluss bekannter Patientenmutationen des SMN1-Gens auf die Stöchiometrie des SMNKomplexes zu untersuchen, sowie den Unterschied zwischen SMN-Komplexen des Cytoplasmas und des Zellkerns zu bestimmen. Dieses Projekt kann dazu beitragen sowohl die Assemblierung der U snRNPs besser zu verstehen, als auch neue Zielmoleküle für Diagnose und Therapie von genetischen Krankheiten wie SMA bereitzustellen.Summary The SMN complex is a macromolecular protein entity consisting of nine fixed subunits (SMN, Gemin2-Gemin8, Unrip) and several transient factors (SmB/B’, SmE, SmF, SmG, SmD1-D3). The main function of this housekeeping complex comprises the assembly of U snRNPs in the cytoplasm and their export to the nucleus where they build up the spliceosome. As a result of mutations in the SMN1 gene this process can be defective and elicits Spinal Muscular Atrophy (SMA). SMA is the second most common autosomal recessive hereditary disease beside cystic fibrosis resulting in death. It leads to a degeneration of the -motor neurons of the spinal cord and subsequently to muscular weakness and wasting. The aim of this study was the characterization of the human Survival of Motor Neuron (SMN) complex composition and stoichiometry by mass spectrometry. In this context, the native stoichiometric composition of the SMN core complex was compared with two recombinant complexes, each incorporating a known mutation (Y272C, E134K) involved in SMA. For this purpose wildtype SMN and mutated versions thereof were expressed as GST-fusion proteins together with full length Gemin2, Gemin6, Gemin7 and Gemin8 in E.coli. After harvesting and lysis of the cells the SMN complex was enriched on a glutathione-sepharose resin and further prepared by carbamidomethylation and tryptic digestion for a quantitaive massspectrometric analysis. Furthermore, the differences between the cytoplasmic and the nuclear SMN complex both derived from nuclei-cytoplasm separation of HeLa cells by a modified Roeder protocol and subsequent Co-immunoprecipitations against the SMN-specific antibody 7B19 were examined . In order to quantify the SMN core complex absolute quantification (AQUA) was applied. Stableisotope labeled peptide analogs were spiked to the native sample in defined amounts thus introducing a mass difference of 6-10 Da during the massspectrometric analysis. Preliminarily, appropriate peptides that meet several conditions (e.g. no labile amino acids, no missed tryptic digestion sites) to allow for a confident quantification were chosen. As MS-analysis was performed by Selected Reaction Monitoring (S/MRM) on two different triple quadrupol systems, transitions and parameters (Declustering Potential and Collision Energy) for each respective peptide were optimized . Alternatively, label-free quantitation on the LTQ Orbitrap Velos was applied. The high mass accuracy and sensitivity of this hybrid FT MS allows quantifying the respective parent ions directly from the MS1 trace. Both strategies, S/MRM and MS1, provide a linear quantification of peptides down to a concentration of 250 amol. By optimizing the current workflow, it was possible to determine the stoichiometry of SMN-complexes containing several known patient mutations within the SMN1 gene as well as discovering the differences between SMN-complexes originating either from cytoplasm or from nucleus. This project may help to throw a light on the assembly of U snRNPs and provide new targets in diagnosis and therapy for genetic diseases like SMA