RESUMO
The childhood disorder spinal muscular atrophy (SMA) is caused by reduced expression of the survival motor neuron (SMN) protein. SMN is a multifunctional protein that has been implicated in the production, processing and transport of RNA and ribonucleoproteins (RNPs). Within the nucleus, SMN is predominantly targeted to Cajal bodies (CB), which are involved in the maturation and processing of several subclasses of RNPs. Here, we show that the SMN exon 2b-encoded domain (SMN2b) is independently sufficient to mediate CB targeting, but that the resulting bodies are less dynamic than those containing full-length SMN protein. We also show that while two SMN proteins harbouring SMA-causing point mutations (A2G and S262I) are efficiently targeted to CBs, they also display reduced nuclear movement.
Assuntos
Corpos Enovelados/metabolismo , Mutação , Proteínas do Complexo SMN/genética , Criança , Éxons , Células HeLa , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Complexo SMN/metabolismoRESUMO
Childhood spinal muscular atrophy is caused by a reduced expression of the survival motor neuron (SMN) protein. SMN has been implicated in the axonal transport of beta-actin mRNA in both primary and transformed neuronal cell lines, and loss of this function could account, at least in part, for spinal muscular atrophy onset and pathological specificity. Here we have utilised a targeted screen to identify mRNA associated with SMN, Gemin2 and Gemin3 in the cytoplasm of a human neuroblastoma cell line, SHSY5Y. Importantly, we have provided the first direct evidence that beta-actin mRNA is present in SMN cytoplasmic complexes in SHSY5Y cells.
Assuntos
Actinas/genética , Citoplasma/metabolismo , Proteína DEAD-box 20/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Axônios , Linhagem Celular Tumoral , HumanosRESUMO
Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca(2+) homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca(2)(+)-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.
Assuntos
Apoptose , Calreticulina/metabolismo , Sequência de Bases , Primers do DNA , Células HeLa , Humanos , Células Jurkat , Microscopia de Fluorescência , Fosfatidilserinas/metabolismo , Reação em Cadeia da Polimerase , Frações Subcelulares/metabolismoRESUMO
Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is a ubiquitously expressed house keeping protein that is involved in RNA production and processing. However, although SMN is expressed in every cell type, only the lower motor neurons of the spinal cord are degraded in SMA. It remains unclear why this is the case. Recently, SMN has been linked to the axonal transport of beta-actin mRNA from the cell body down to the growth cones. beta-Actin is transported actively in neurite granules (NGs). However, it remains unclear which known SMN-binding partners are present in these SMN-NGs. To address this we have analysed SMN-NGs in a human neuronal cell line, SH-SY5Y, using antibodies against the majority of reported SMN-binding partners, including: Gemin2, Gemin3, Gemin4, Gemin5, Gemin6, Gemin7, Sm core proteins, fibrillarin, EWS, PFNII, Unrip and ZPR1. The obtained results highlight the metamorphic nature of the SMN complex, suggesting that not all the "core" SMN-binding proteins are transported in SMN-NGs.
Assuntos
Corpos Enovelados/metabolismo , Atrofia Muscular Espinal/metabolismo , Neuritos/metabolismo , Proteínas do Complexo SMN/metabolismo , Vesículas Transportadoras/metabolismo , Pré-Escolar , Citoplasma/metabolismo , Células HeLa , Humanos , Transporte ProteicoRESUMO
Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is expressed in every cell type, but it is predominantly the lower motor neurones of the spinal cord that degenerate in SMA. SMN has been linked to the axonal transport of beta-actin mRNA, a breakdown in which could trigger disease onset. It is known that SMN is present in transport ribonucleoproteins (RNPs) granules that also contain Gemin2 and Gemin3. To further characterise these granules we have performed live cell imaging of GFP-tagged SMN, GFP-Gemin2, GFP-Gemin3, GFP-Gemin6 and GFP-Gemin7. In all, we have made two important observations: (1) SMN granules appear metamorphic; and (2) the SMN-Gemin complex(es) appears to localise to two distinct subsets of bodies in neurites; stationary bodies and smaller dynamic bodies. This study provides an insight into the neuronal function of the SMN complex.
Assuntos
Neuritos/metabolismo , Proteínas do Complexo SMN/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Vesículas Transportadoras/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/metabolismo , Humanos , Atrofias Musculares Espinais da Infância/metabolismoRESUMO
The Ewing's sarcoma (EWS) protein is a ubiquitously expressed RNA chaperone. The EWS protein localizes predominantly to the nucleus. Previous reports have suggested that the EWS protein is capable of dimerizing. However, to date this has not been confirmed. Here, using a novel panel of recombinant proteins, we have performed an in vitro biomolecular interaction analysis of the EWS protein. We have demonstrated that all three arginine-glycine-glycine (RGG) motifs are capable of binding directly to the survival motor neuron protein, a Tudor domain containing EWS binding partner. We have also confirmed EWS is capable of self-associating, and we have mapped this binding domain to the RGG motifs. We have also found that self-association may be required for EWS nuclear import. This is the first direct evidence of RGG domains being involved in self-association and has implications on all RGG-containing proteins.
Assuntos
Proteínas do Tecido Nervoso/química , Multimerização Proteica , Proteína EWS de Ligação a RNA/química , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Arginina/metabolismo , Sítios de Ligação , Cromatografia em Gel , Glicina/metabolismo , Células HeLa , Humanos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ligação Proteica , Proteína EWS de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Proteína 1 de Sobrevivência do Neurônio MotorRESUMO
The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.
Assuntos
Neoplasias Ósseas/metabolismo , Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Dedos de Zinco , Transporte Ativo do Núcleo Celular , Citoplasma/metabolismo , Análise Mutacional de DNA , Humanos , Mutação , Sinais Direcionadores de Proteínas , Proteína EWS de Ligação a RNA/genéticaRESUMO
Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex.
Assuntos
Sinais de Localização Nuclear/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citoplasma/metabolismo , Células HeLa , Humanos , Antígenos de Histocompatibilidade Menor , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Sinais de Localização Nuclear/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio MotorRESUMO
The spliceosome is the RNP complex than catalyses the removal of introns from the Uridine-rich small nuclear ribonucleoproteins (U snRNPs) that make up the main components of this complex. The production of these RNPs is an intricate process, involving several key stages. These include: 1) the transcription of the U snRNAs; 2) their nuclear export; 3) the cytoplasmic assembly of the U snRNP; 4) their nuclear import; 5) their processing within Cajal bodies and the nucleolus; and 6) their storage in interchromatin granule clusters (IGCs). This review focuses on each of these stages, discussing the key complexes involved as well as the trafficking and targeting mechanisms involved.
Assuntos
Nucléolo Celular/metabolismo , Corpos Enovelados/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Splicing de RNA , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Transcrição GênicaRESUMO
The childhood autosomal recessive disorder spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN) gene. SMN localizes diffusely in the cytoplasm and in distinct nuclear structures called Cajal bodies. Cajal bodies are believed to be the storage and processing sites of several ribonucleoproteins. Here, using a novel panel of SMN exon deletion constructs, we report a systematic analysis of internal targeting domains in the SMN protein. We demonstrate that the peptides encoded by exons 2b, 3 and 6 perform an integral role in the cellular targeting of SMN. In addition, we identify a nine amino acid motif within the highly conserved sequences of the exon 2b encoded domain that mediates Cajal body targeting and self-association. Deletion of this domain dramatically affects SMN activity and results in a dominant-negative clone. These results identify critical domains within the SMN protein and have an impact on our understanding of the SMN protein with regards to SMA as well as cellular biology.