scholarly journals Solution structure of the core SMN–Gemin2 complex

2012 ◽  
Vol 445 (3) ◽  
pp. 361-370 ◽  
Author(s):  
Kathryn L. Sarachan ◽  
Kathleen G. Valentine ◽  
Kushol Gupta ◽  
Veronica R. Moorman ◽  
John M. Gledhill ◽  
...  
Keyword(s):  
Solution Structure ◽  
Muscular Atrophy ◽  
Small Nuclear Rna ◽  
Sm Proteins ◽  
Binding Interface ◽  
Smn Complex ◽  
Nuclear Rna ◽  
Smn Protein ◽  
Survival Of Motor Neuron ◽  
Small Nuclear Ribonucleoproteins

In humans, assembly of spliceosomal snRNPs (small nuclear ribonucleoproteins) begins in the cytoplasm where the multi-protein SMN (survival of motor neuron) complex mediates the formation of a seven-membered ring of Sm proteins on to a conserved site of the snRNA (small nuclear RNA). The SMN complex contains the SMN protein Gemin2 and several additional Gemins that participate in snRNP biosynthesis. SMN was first identified as the product of a gene found to be deleted or mutated in patients with the neurodegenerative disease SMA (spinal muscular atrophy), the leading genetic cause of infant mortality. In the present study, we report the solution structure of Gemin2 bound to the Gemin2-binding domain of SMN determined by NMR spectroscopy. This complex reveals the structure of Gemin2, how Gemin2 binds to SMN and the roles of conserved SMN residues near the binding interface. Surprisingly, several conserved SMN residues, including the sites of two SMA patient mutations, are not required for binding to Gemin2. Instead, they form a conserved SMN/Gemin2 surface that may be functionally important for snRNP assembly. The SMN–Gemin2 structure explains how Gemin2 is stabilized by SMN and establishes a framework for structure–function studies to investigate snRNP biogenesis as well as biological processes involving Gemin2 that do not involve snRNP assembly.

scholarly journals Regulation of SMN Protein Stability

2008 ◽  
Vol 29 (5) ◽  
pp. 1107-1115 ◽  
Author(s):  
Barrington G. Burnett ◽  
Eric Muñoz ◽  
Animesh Tandon ◽  
Deborah Y. Kwon ◽  
Charlotte J. Sumner ◽  
...  
Keyword(s):  
Complex Formation ◽  
Protein Stability ◽  
Half Life ◽  
Muscular Atrophy ◽  
Ubiquitin Proteasome System ◽  
Protein Product ◽  
Smn Complex ◽  
A Cell ◽  
Smn Protein ◽  
Survival Of Motor Neuron

ABSTRACT Spinal muscular atrophy (SMA) is caused by mutations of the survival of motor neuron (SMN1) gene and deficiency of full-length SMN protein (FL-SMN). All SMA patients retain one or more copies of the SMN2 gene, but the principal protein product of SMN2 lacks exon 7 (SMNΔ7) and is unable to compensate for a deficiency of FL-SMN. SMN is known to oligomerize and form a multimeric protein complex; however, the mechanisms regulating stability and degradation of FL-SMN and SMNΔ7 proteins have been largely unexplored. Using pulse-chase analysis, we characterized SMN protein turnover and confirmed that SMN was ubiquitinated and degraded by the ubiquitin proteasome system (UPS). The SMNΔ7 protein had a twofold shorter half-life than FL-SMN in cells despite similar intrinsic rates of turnover by the UPS in a cell-free assay. Mutations that inhibited SMN oligomerization and complex formation reduced the FL-SMN half-life. Furthermore, recruitment of SMN into large macromolecular complexes as well as increased association with several Gemin proteins was regulated in part by protein kinase A. Together, our data indicate that SMN protein stability is modulated by complex formation. Promotion of the SMN complex formation may be an important novel therapeutic strategy for SMA.

scholarly journals Interaction between the small‐nuclear‐RNA cap hypermethylase and the spinal muscular atrophy protein, survival of motor neuron

EMBO Reports ◽  
2003 ◽  
Vol 4 (6) ◽  
pp. 616-622 ◽  
Author(s):  
John Mouaikel ◽  
Usha Narayanan ◽  
Céline Verheggen ◽  
A Gregory Matera ◽  
Edouard Bertrand ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Muscular Atrophy ◽  
Small Nuclear Rna ◽  
Nuclear Rna ◽  
Survival Of Motor Neuron

scholarly journals Composition of the Survival Motor Neuron (SMN) complex in Drosophila melanogaster

2018 ◽  
Author(s):  
A. Gregory Matera ◽  
Amanda C. Raimer ◽  
Casey A. Schmidt ◽  
Jo A. Kelly ◽  
Gaith N. Droby ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Motor Neuron ◽  
Muscular Atrophy ◽  
Structural Similarity ◽  
Survival Motor Neuron ◽  
Sm Proteins ◽  
Native Promoter ◽  
Smn Complex ◽  
Biological Studies ◽  
Smn Protein

AbstractSpinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated the composition of the SMN complex in Drosophila melanogaster. Using a stable transgenic line that exclusively expresses Flag-tagged SMN from its native promoter, we previously found that Gemin2, Gemin3, Gemin5, and all nine classical Sm proteins, including Lsm10 and Lsm11, co-purify with SMN. Here, we show that CG2941 is also highly enriched in the pulldown. Reciprocal co-immunoprecipitation reveals that epitope-tagged CG2941 interacts with endogenous SMN in Schneider2 cells. Bioinformatic comparisons show that CG2941 shares sequence and structural similarity with metazoan Gemin4. Additional analysis shows that three other genes (CG14164, CG31950 and CG2371) are not orthologous to Gemins 6-7-8, respectively, as previously suggested. In D.melanogaster, CG2941 is located within an evolutionarily recent genomic triplication with two other nearly identical paralogous genes (CG32783 and CG32786). RNAi-mediated knockdown of CG2941 and its two close paralogs reveals that Gemin4 is essential for organismal viability.

scholarly journals The SMN Complex Is Associated with snRNPs throughout Their Cytoplasmic Assembly Pathway

2002 ◽  
Vol 22 (18) ◽  
pp. 6533-6541 ◽  
Author(s):  
Séverine Massenet ◽  
Livio Pellizzoni ◽  
Sergey Paushkin ◽  
Iain W. Mattaj ◽  
Gideon Dreyfuss
Keyword(s):  
Motor Neurons ◽  
Muscular Atrophy ◽  
Sm Proteins ◽  
Assembly Pathway ◽  
Smn Complex ◽  
Complex Functions ◽  
Survival Of Motor Neurons ◽  
The Common ◽  
Smn Protein ◽  
Cytoplasmic Assembly

ABSTRACT The common neurodegenerative disease spinal muscular atrophy is caused by reduced levels of the survival of motor neurons (SMN) protein. SMN associates with several proteins (Gemin2 to Gemin6) to form a large complex which is found both in the cytoplasm and in the nucleus. The SMN complex functions in the assembly and metabolism of several RNPs, including spliceosomal snRNPs. The snRNP core assembly takes place in the cytoplasm from Sm proteins and newly exported snRNAs. Here, we identify three distinct cytoplasmic SMN complexes, each representing a defined intermediate in the snRNP biogenesis pathway. We show that the SMN complex associates with newly exported snRNAs containing the nonphosphorylated form of the snRNA export factor PHAX. The second SMN complex identified contains assembled Sm cores and m3G-capped snRNAs. Finally, the SMN complex is associated with a preimport complex containing m3G-capped snRNP cores bound to the snRNP nuclear import mediator snurportin1. Thus, the SMN complex is associated with snRNPs during the entire process of their biogenesis in the cytoplasm and may have multiple functions throughout this process.

scholarly journals Sumoylation regulates the assembly and activity of the SMN complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Giulietta M. Riboldi ◽  
Irene Faravelli ◽  
Takaaki Kuwajima ◽  
Nicolas Delestrée ◽  
Georgia Dermentzaki ◽  
...  
Keyword(s):  
Survival Rate ◽  
Muscular Atrophy ◽  
Motor Deficits ◽  
Cellular Distribution ◽  
Post Translational Modification ◽  
Smn Complex ◽  
Sensory Motor ◽  
Motor Neuron Loss ◽  
And Function ◽  
Small Nuclear Ribonucleoproteins

AbstractSMN is a ubiquitously expressed protein and is essential for life. SMN deficiency causes the neurodegenerative disease spinal muscular atrophy (SMA), the leading genetic cause of infant mortality. SMN interacts with itself and other proteins to form a complex that functions in the assembly of ribonucleoproteins. SMN is modified by SUMO (Small Ubiquitin-like Modifier), but whether sumoylation is required for the functions of SMN that are relevant to SMA pathogenesis is not known. Here, we show that inactivation of a SUMO-interacting motif (SIM) alters SMN sub-cellular distribution, the integrity of its complex, and its function in small nuclear ribonucleoproteins biogenesis. Expression of a SIM-inactivated mutant of SMN in a mouse model of SMA slightly extends survival rate with limited and transient correction of motor deficits. Remarkably, although SIM-inactivated SMN attenuates motor neuron loss and improves neuromuscular junction synapses, it fails to prevent the loss of sensory-motor synapses. These findings suggest that sumoylation is important for proper assembly and function of the SMN complex and that loss of this post-translational modification impairs the ability of SMN to correct selective deficits in the sensory-motor circuit of SMA mice.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction.

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577 ◽  
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

scholarly journals U4 small nuclear RNA dissociates from a yeast spliceosome and does not participate in the subsequent splicing reaction

1991 ◽  
Vol 11 (11) ◽  
pp. 5571-5577
Author(s):  
S L Yean ◽  
R J Lin
Keyword(s):  
Mrna Splicing ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Small Nuclear Rna ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Sensitive Allele ◽  
U4 Rna ◽  
Small Nuclear Ribonucleoproteins

U4 and U6 small nuclear RNAs reside in a single ribonucleoprotein particle, and both are required for pre-mRNA splicing. The U4/U6 and U5 small nuclear ribonucleoproteins join U1 and U2 on the pre-mRNA during spliceosome assembly. Binding of U4 is then destabilized prior to or concomitant with the 5' cleavage-ligation. In order to test the role of U4 RNA, we isolated a functional spliceosome by using extracts prepared from yeast cells carrying a temperature-sensitive allele of prp2 (rna2). The isolated prp2 delta spliceosome contains U2, U5, U6, and possibly also U1 and can be activated to splice the bound pre-mRNA. U4 RNA does not associate with the isolated spliceosomes and is shown not to be involved in the subsequent cleavage-ligation reactions. These results are consistent with the hypothesis that the role of U4 in pre-mRNA splicing is to deliver U6 to the spliceosome.

Splicing efficiency of minor introns in a mouse model of SMA predominantly depends on their branchpoint sequence and can involve the contribution of major spliceosome components.

RNA ◽  
2021 ◽  
pp. rna.078329.120
Author(s):  
Valentin Jacquier ◽  
Manon Prevot ◽  
Thierry Gostan ◽  
Remy Bordonne ◽  
Sofia Benkhelifa-Ziyyat ◽  
...  
Keyword(s):  
Muscular Atrophy ◽  
Differential Splicing ◽  
Molecular Determinants ◽  
In Vitro Splicing ◽  
Splicing Efficiency ◽  
Smn Protein ◽  
Survival Of Motor Neuron ◽  
Branchpoint Sequence ◽  
First Time

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease caused by reduced amounts of the ubiquitously expressed Survival of Motor Neuron (SMN) protein. In agreement with its crucial role in the biogenesis of spliceosomal snRNPs, SMN-deficiency is correlated to numerous splicing alterations in patient cells and various tissues of SMA mouse models. Among the snRNPs whose assembly is impacted by SMN-deficiency, those involved in the minor spliceosome are particularly affected. Importantly, splicing of several, but not all U12-dependent introns has been shown to be affected in different SMA models. Here, we have investigated the molecular determinants of this differential splicing in spinal cords from SMA mice. We show that the branchpoint sequence (BPS) is a key element controlling splicing efficiency of minor introns. Unexpectedly, splicing of several minor introns with suboptimal BPS is not affected in SMA mice. Using in vitro splicing experiments and oligonucleotides targeting minor or major snRNAs, we show for the first time that splicing of these introns involves both the minor and major machineries. Our results strongly suggest that splicing of a subset of minor introns is not affected in SMA mice because components of the major spliceosome compensate for the loss of minor splicing activity.

scholarly journals The Survival of Motor Neurons Protein Determines the Capacity for snRNP Assembly: Biochemical Deficiency in Spinal Muscular Atrophy

2005 ◽  
Vol 25 (13) ◽  
pp. 5543-5551 ◽  
Author(s):  
Lili Wan ◽  
Daniel J. Battle ◽  
Jeongsik Yong ◽  
Amelie K. Gubitz ◽  
Stephen J. Kolb ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neurons ◽  
Muscular Atrophy ◽  
Protein Levels ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Smn Complex ◽  
Survival Of Motor Neurons ◽  
Smn Protein ◽  
Nuclear Ribonucleoprotein

ABSTRACT 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.

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