RESUMO
Spinal muscular atrophy is caused by reduced levels of SMN resulting from the loss of SMN1 and reliance on SMN2 for the production of SMN. Loss of SMN entirely is embryonic lethal in mammals. There are several SMN missense mutations found in humans. These alleles do not show partial function in the absence of wild-type SMN and cannot rescue a null Smn allele in mice. However, these human SMN missense allele transgenes can rescue a null Smn allele when SMN2 is present. We find that the N- and C-terminal regions constitute two independent domains of SMN that can be separated genetically and undergo intragenic complementation. These SMN protein heteromers restore snRNP assembly of Sm proteins onto snRNA and completely rescue both survival of Smn null mice and motor neuron electrophysiology demonstrating that the essential functional unit of SMN is the oligomer.
Assuntos
Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Alelos , Aminoácidos/genética , Animais , Modelos Animais de Doenças , Éxons/genética , Predisposição Genética para Doença , Humanos , Camundongos , Camundongos Knockout , Neurônios Motores/patologia , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Mutação de Sentido Incorreto/genética , Multimerização Proteica/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN/genéticaRESUMO
Reduced expression of SMN causes spinal muscular atrophy, a severe neurodegenerative disease. Despite the importance of maintaining SMN levels, relatively little is known about the mechanisms by which SMN levels are regulated. We show here that Gemin5, the snRNA-binding protein of the SMN complex, binds directly to the SMN mRNA and regulates SMN expression. Gemin5 binds with high specificity, both in vitro and in vivo, to sequence and structural elements in the SMN mRNA 3'-untranslated region that are reminiscent of the snRNP code to which Gemin5 binds on snRNAs. Reduction of Gemin5 redistributes the SMN mRNA from heavy polysomes to lighter polysomes and monosomes, suggesting that Gemin5 functions as an activator of SMN translation. SMN protein is not stoichiometrically present on the SMN mRNA with Gemin5, but the mRNA-binding activity of Gemin5 is dependent on SMN levels, providing a feedback mechanism for SMN to regulate its own expression via Gemin5. This work both reveals a new autoregulatory pathway governing SMN expression, and identifies a new mechanism through which SMN can modulate specific mRNA expression via Gemin5.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/biossíntese , Animais , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Polirribossomos/genética , Polirribossomos/metabolismo , Ligação Proteica , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Insufficient expression of the survival motor neuron (SMN) protein causes spinal muscular atrophy, a neurodegenerative disease characterized by loss of motor neurons. Despite the importance of maintaining adequate SMN levels, little is known about factors that control SMN expression, particularly 3' end processing of the SMN pre-mRNA. In this study, we identify the U1A protein as a key regulator of SMN expression. U1A, a component of the U1 snRNP, is known to inhibit polyadenylation upon direct binding to mRNA. We show that U1A binds directly and with high affinity and specificity to the SMN 3'-UTR adjacent to the polyadenylation site, independent of the U1 snRNP (U1 small nuclear ribonucleoprotein). Binding of U1A inhibits polyadenylation of the SMN pre-mRNA by specifically inhibiting 3' cleavage by the cleavage and polyadenylation specificity factor. Expression of U1A in excess of U1 snRNA causes inhibition of SMN polyadenylation and decreases SMN protein levels. This work reveals a new mechanism for regulating SMN levels and provides new insight into the roles of U1A in 3' processing of mRNAs.
Assuntos
Regiões 3' não Traduzidas/fisiologia , Poliadenilação/fisiologia , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Células 3T3 , Animais , Células HeLa , Humanos , Camundongos , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genéticaRESUMO
Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN, together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels, as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known. To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts. Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a biochemical activity in cells from patients with spinal muscular atrophy.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Biotinilação , Extratos Celulares/análise , Linhagem Celular , Linhagem Celular Transformada , Transformação Celular Viral , Galinhas , Citoplasma/química , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HeLa , Herpesvirus Humano 4 , Humanos , Cinética , Modelos Biológicos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/patologia , Radioisótopos de Fósforo , Ligação Proteica , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/análise , Proteínas do Complexo SMN , Sensibilidade e Especificidade , Transcrição GênicaRESUMO
The lymphotropic Herpesvirus saimiri (HVS) causes acute leukemia, T-cell lymphoma, and death in New World monkeys. HVS encodes seven small RNAs (HSURs) of unknown function. The HSURs acquire host Sm proteins and assemble Sm cores similar to those found on the spliceosomal small nuclear RNPs (snRNPs). Here we show that, like host snRNPs, HSURs use the SMN (survival of motor neurons) complex to assemble Sm cores. The HSURs bind the SMN complex directly and with very high affinity, similar to or higher than that of host snRNAs, and can outcompete host snRNAs for SMN-dependent assembly into RNPs. These observations highlight the general utility of the SMN complex for RNP assembly and suggest that infectious agents that engage the SMN complex may burden SMN-dependent pathways, possibly leading to a deleterious reduction in available SMN complex for essential host functions.
Assuntos
Herpesvirus Saimiriíneo 2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA Nuclear Pequeno/metabolismo , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Genes Reporter , Células HeLa , Herpesvirus Saimiriíneo 2/genética , Humanos , Complexos Multiproteicos , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Nuclear Pequeno/genética , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMNRESUMO
To serve in its function as an assembly machine for spliceosomal small nuclear ribonucleoprotein particles (snRNPs), the survival of motor neurons (SMN) protein complex binds directly to the Sm proteins and the U snRNAs. A specific domain unique to U1 snRNA, stem-loop 1 (SL1), is required for SMN complex binding and U1 snRNP Sm core assembly. Here, we show that each of the major spliceosomal U snRNAs (U2, U4, and U5), as well as the minor splicing pathway U11 snRNA, contains a domain to which the SMN complex binds directly and with remarkable affinity (low nanomolar concentration). The SMN-binding domains of the U snRNAs do not have any significant nucleotide sequence similarity yet they compete for binding to the SMN complex in a manner that suggests the presence of at least two binding sites. Furthermore, the SMN complex-binding domain and the Sm site are both necessary and sufficient for Sm core assembly and their relative positions are critical for snRNP assembly. These findings indicate that the SMN complex stringently scrutinizes RNAs for specific structural features that are not obvious from the sequence of the RNAs but are required for their identification as bona fide snRNAs. It is likely that this surveillance capacity of the SMN complex ensures assembly of Sm cores on the correct RNAs only and prevents illicit, potentially deleterious, assembly of Sm cores on random RNAs.
Assuntos
RNA Nuclear Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Autoantígenos , Células HeLa , Humanos , Substâncias Macromoleculares , Neurônios Motores/metabolismo , Conformação de Ácido Nucleico , Oócitos/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína , Ribonucleoproteínas Nucleares Pequenas/genética , Spliceossomos/metabolismo , Xenopus laevis , Proteínas Centrais de snRNPRESUMO
The SMN complex is essential for the biogenesis of spliceosomal small nuclear ribonucleoproteins and likely functions in the assembly, metabolism, and transport of a diverse number of other ribonucleoproteins. Specifically, the SMN complex assembles 7 Sm proteins into a core structure around a highly conserved sequence of ribonucleic acid (RNA) found in small nuclear RNAs. The complex recognizes specific sequences and structural features of small nuclear RNAs and Sm proteins and assembles small nuclear ribonucleoproteins in a stepwise fashion. In addition to the SMN protein, the SMN complex contains 7 additional proteins known as Gemin2-8, each likely to play a role in ribonucleoprotein biogenesis. This review focuses on the current understanding of the mechanism of the role of the SMN complex in small nuclear ribonucleoprotein assembly and considers the relationship of this function to spinal muscular atrophy.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Ribonucleoproteínas Nucleares Pequenas/genética , Atrofias Musculares Espinais da Infância/genética , Criança , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Substâncias Macromoleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , RNA/biossíntese , RNA/genética , Splicing de RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas do Complexo SMN , Atrofias Musculares Espinais da Infância/metabolismo , Atrofias Musculares Espinais da Infância/fisiopatologiaRESUMO
The SMN protein is essential and participates in the assembly of macromolecular complexes of RNA and protein in all cells. The best-characterized function of SMN is as an assembler of spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN performs this function as part of a complex with several other proteins called Gemins. snRNPs are assembled in the cytoplasm in a stepwise manner and then are imported to the nucleus where they participate globally in the splicing of pre-mRNA. Mutations in the SMN1 gene result in the motor neuron disease, spinal muscular atrophy (SMA). Most of these mutations result in a reduction in the expression levels of the SMN protein, which, in turn, results in a reduction in snRNP assembly capacity. This review highlights current studies that have investigated the mechanism of SMN-dependent snRNP assembly, as well as the downstream effects on pre-mRNA splicing that result from a decrease in SMN. This article is part of a Special Issue entitled "RNA-Binding Proteins".
Assuntos
Doença dos Neurônios Motores/genética , Neurônios Motores/fisiologia , Atrofia Muscular Espinal/genética , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Animais , Humanos , Doença dos Neurônios Motores/fisiopatologia , Atrofia Muscular Espinal/fisiopatologia , Mutação/genética , Mutação/fisiologia , Ribonucleoproteínas Nucleares Pequenas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Proteína 1 de Sobrevivência do Neurônio Motor/fisiologiaRESUMO
The survival of motor neurons (SMN) complex is essential for the biogenesis of small nuclear ribonucleoprotein (snRNP) complexes in eukaryotic cells. Reduced levels of SMN cause the motor neuron degenerative disease, spinal muscular atrophy. We identify here stable subunits of the SMN complex that do not contain SMN. Sedimentation and immunoprecipitation experiments using cell extracts reveal at least three complexes composed of Gemin3, -4, and -5; Gemin6, -7, and unrip; and SMN with Gemin2, as well as free Gemin5. Complexes containing Gemin3-Gemin4-Gemin5 and Gemin6-Gemin7-unrip persist at similar levels when SMN is reduced. In cells, immunofluorescence microscopy shows differential localization of Gemin5 after cell stress. We further show that the Gemin5-containing subunits bind small nuclear RNA independently of the SMN complex and without a requirement for exogenous ATP. ATP hydrolysis is, however, required for displacement of small nuclear RNAs from the Gemin5-containing subunits and their assembly into snRNPs. These findings demonstrate a modular nature of the SMN complex and identify a new intermediate in the snRNP assembly process.
Assuntos
Ribonucleoproteínas Nucleares Pequenas/fisiologia , Trifosfato de Adenosina/química , Células HeLa , Temperatura Alta , Humanos , Hidrólise , Imunoprecipitação , Microscopia de Fluorescência/métodos , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Interferência de RNA , RNA Nuclear Pequeno/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , Sacarose/química , Sacarose/metabolismo , Sacarose/farmacologia , Fatores de TempoRESUMO
The survival of motor neurons protein (SMN) is part of a large complex that contains six other proteins, Gemins2-7. The SMN complex assembles the heptameric Sm protein core on small nuclear RNAs (snRNAs) and plays a critical role in the biogenesis of snRNPs, the major and essential components of mRNA splicing in eukaryotes. For its function, the SMN complex binds Sm proteins and snRNAs, which it distinguishes from other RNAs by specific features they contain. We show here that Gemin5, a 170 kDa WD-repeat protein, is the snRNA binding protein of the SMN complex. Gemin5 binds directly and specifically to the unique features, including the Sm site, of snRNAs. Reduction of Gemin5 results in reduced capacity of the SMN complex to bind snRNAs and to assemble Sm cores. Gemin5 therefore functions as the factor that allows the SMN complex to distinguish snRNAs from other cellular RNAs for snRNP biogenesis.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Células Cultivadas , Células HeLa , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMNRESUMO
Reduction in the expression of the survival of motor neurons (SMN) protein results in spinal muscular atrophy (SMA), a common motor neuron degenerative disease. SMN is part of a large macromolecular complex (the SMN complex) that includes at least six additional proteins called Gemins (Gemin2-7). The SMN complex is expressed in all cells and is present throughout the cytoplasm and in the nucleus where it is concentrated in Gems. The SMN complex plays an essential role in the production of spliceosomal small nuclear ribonucleoproteins (snRNPs) and likely other RNPs. To study the roles of the individual proteins, we systematically reduced the expression of SMN and each of the Gemins (2-6) by RNA interference. We show that the reduction of SMN leads to a decrease in snRNP assembly, the disappearance of Gems, and to a drastic reduction in the amounts of several Gemins. Moreover, reduction of Gemin2 or Gemin6 strongly decreases the activity of the SMN complex. These findings demonstrate that other components of the SMN complex, in addition to SMN, are critical for the activity of the complex and suggest that Gemin2 and Gemin6 are potentially important modifiers of SMA as well as potential disease genes for non-SMN motor neuron diseases.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica , Células HeLa/metabolismo , Humanos , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , TransfecçãoRESUMO
Functional RNAs often form compact structures characterized by closely packed helices. Crystallographic analysis of several large RNAs revealed a prevalent interaction in which unpaired adenosine residues dock into the minor groove of a receptor helix. This A-minor motif, potentially the most important element responsible for global RNA architecture, has also been suggested to contribute to the fidelity of protein synthesis by discriminating against near-cognate tRNAs on the ribosome. The specificity of A-minor interactions is fundamental to RNA tertiary structure formation, as well as to their proposed role in translational accuracy. To investigate A-minor motif specificity, we analyzed mutations in an A-minor interaction within the Tetrahymena group I self-splicing intron. Thermodynamic and x-ray crystallographic results show that the A-minor interaction strongly prefers canonical base pairs over base mismatches in the receptor helix, enabling RNA interhelical packing through specific recognition of Watson-Crick minor groove geometry.