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 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 Identification of Novel Compounds That Increase SMN Protein Levels Using an Improved SMN2 Reporter Cell Assay

2012 ◽  
Vol 17 (4) ◽  
pp. 481-495 ◽  
Author(s):  
Jonathan J. Cherry ◽  
Matthew C. Evans ◽  
Jake Ni ◽  
Gregory D. Cuny ◽  
Marcie A. Glicksman ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Neurodegenerative Disorder ◽  
Muscular Atrophy ◽  
Full Length ◽  
Reporter Cell ◽  
Protein Levels ◽  
Smn2 Gene ◽  
Smn Protein ◽  
Neuron Function ◽  
Survival Of Motor Neuron

Spinal muscular atrophy (SMA) is a neurodegenerative disorder that is characterized by progressive loss of motor neuron function. It is caused by the homozygous loss of the SMN1 ( survival of motor neuron 1) gene and a decrease in full-length SMN protein. SMN2 is a nearly identical homolog of SMN1 that, due to alternative splicing, expresses predominantly truncated SMN protein. SMN2 represents an enticing therapeutic target. Increasing expression of full-length SMN from the SMN2 gene might represent a treatment for SMA. We describe a newly designed cell-based reporter assay that faithfully and reproducibly measures full-length SMN expression from the SMN2 gene. This reporter can detect increases of SMN protein by an array of compounds previously shown to regulate SMN2 expression and by the overexpression of proteins that modulate SMN2 splicing. It also can be used to evaluate changes at both the transcriptional and splicing level. This assay can be a valuable tool for the identification of novel compounds that increase SMN2 protein levels and the optimization of compounds already known to modulate SMN2 expression. We present here preliminary data from a high-throughput screen using this assay to identify novel compounds that increase expression of SMN2.

scholarly journals The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein

2013 ◽  
Vol 24 (12) ◽  
pp. 1863-1871 ◽  
Author(s):  
Deborah Y. Kwon ◽  
Maria Dimitriadi ◽  
Barbara Terzic ◽  
Casey Cable ◽  
Anne C. Hart ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Ubiquitin Ligase ◽  
Cultured Cells ◽  
Muscular Atrophy ◽  
E3 Ubiquitin Ligase ◽  
Proteasome Inhibition ◽  
Protein Levels ◽  
Smn Protein ◽  
Survival Of Motor Neuron

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.

In Vitro Restoration of Functional SMN Protein in Human Trophoblast Cells Affected by Spinal Muscular Atrophy by Small Fragment Homologous Replacement

2005 ◽  
Vol 0 (0) ◽  
pp. 050701034702010
Author(s):  
Federica Sangiuolo ◽  
Antonio Filareto ◽  
Paola Spitalieri ◽  
Maria Lucia Scaldaferri ◽  
Ruggiero Mango ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Small Fragment ◽  
Trophoblast Cells ◽  
Human Trophoblast ◽  
Smn Protein ◽  
Human Trophoblast Cells

Spinal Muscular Atrophy

2017 ◽  
Author(s):  
Umrao R. Monani ◽  
Darryl C. De Vivo
Keyword(s):  
Infant Mortality ◽  
Life Expectancy ◽  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Carrier Frequency ◽  
Muscular Atrophy ◽  
Good Treatment ◽  
Motor Neuron Disorder ◽  
Smn Protein ◽  
Survival Of Motor Neuron

Spinal muscular atrophy (SMA) is a common, inherited, pediatric motor neuron disorder caused by insufficient SMN protein. As of yet, there is no good treatment for the disease. SMA has an incidence of ~1 in 10,000 newborns carrier frequency of 1 in 50, making it the most common inherited cause of infant mortality. Patients with severe SMA, or Werdnig-Hoffman disease, typically manifest weakness during the first 6 months of life. Such patients are so debilitated that they never sit independently, frequently succumbing to the disease before age 2 years. A much milder form of SMA, Kugelberg-Welander disease, with onset after 18 months of age, often during childhood and characterized by prolonged ambulation and a normal life expectancy, was described in 1956. In 1995 mutations in a novel gene, Survival of Motor Neuron 1 (SMN1), were determined to be the specific cause of SMA.

scholarly journals Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Melissa B. Walsh ◽  
Eva Janzen ◽  
Emily Wingrove ◽  
Seyyedmohsen Hosseinibarkooie ◽  
Natalia Rodriguez Muela ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Motor Neuron ◽  
Muscular Atrophy ◽  
Protein Complexes ◽  
Genetic Modifiers ◽  
Functional Connection ◽  
C Elegans ◽  
Hnrnp F ◽  
Smn Protein ◽  
Survival Of Motor Neuron

Abstract Background Understanding the genetic modifiers of neurodegenerative diseases can provide insight into the mechanisms underlying these disorders. Here, we examine the relationship between the motor neuron disease spinal muscular atrophy (SMA), which is caused by reduced levels of the survival of motor neuron (SMN) protein, and the actin-bundling protein Plastin 3 (PLS3). Increased PLS3 levels suppress symptoms in a subset of SMA patients and ameliorate defects in SMA disease models, but the functional connection between PLS3 and SMN is poorly understood. Results We provide immunohistochemical and biochemical evidence for large protein complexes localized in vertebrate motor neuron processes that contain PLS3, SMN, and members of the hnRNP F/H family of proteins. Using a Caenorhabditis elegans (C. elegans) SMA model, we determine that overexpression of PLS3 or loss of the C. elegans hnRNP F/H ortholog SYM-2 enhances endocytic function and ameliorates neuromuscular defects caused by decreased SMN-1 levels. Furthermore, either increasing PLS3 or decreasing SYM-2 levels suppresses defects in a C. elegans ALS model. Conclusions We propose that hnRNP F/H act in the same protein complex as PLS3 and SMN and that the function of this complex is critical for endocytic pathways, suggesting that hnRNP F/H proteins could be potential targets for therapy development.

scholarly journals Characterization of SRp46, a Novel Human SR Splicing Factor Encoded by a PR264/SC35 Retropseudogene

1998 ◽  
Vol 18 (8) ◽  
pp. 4924-4934 ◽  
Author(s):  
Johann Soret ◽  
Renata Gattoni ◽  
Cécile Guyon ◽  
Alain Sureau ◽  
Michel Popielarz ◽  
...  
Keyword(s):  
Splicing Factor ◽  
Sr Protein ◽  
Adenovirus E1a ◽  
Cdna Sequences ◽  
Normal Tissues ◽  
Protein Functions ◽  
S100 Extract ◽  
In Vitro Splicing ◽  
First Time

ABSTRACT The highly conserved SR family contains a growing number of phosphoproteins acting as both essential and alternative splicing factors. In this study, we have cloned human genomic and cDNA sequences encoding a novel SR protein designated SRp46. Nucleotide sequence analyses have revealed that the SRp46 gene corresponds to an expressed PR264/SC35 retropseudogene. As a result of mutations and amplifications, the SRp46 protein significantly differs from the PR264/SC35 factor, mainly at the level of its RS domain. Northern and Western blot analyses have established that SRp46 sequences are expressed at different levels in several human cell lines and normal tissues, as well as in simian cells. In contrast, sequences homologous to SRp46 are not present in mice. In vitro splicing studies indicate that the human SRp46 recombinant protein functions as an essential splicing factor in complementing a HeLa cell S100 extract deficient in SR proteins. In addition, complementation analyses performed with β-globin or adenovirus E1A transcripts and different splicing-deficient extracts have revealed that SRp46 does not display the same activity as PR264/SC35. These results demonstrate, for the first time, that an SR splicing factor, which represents a novel member of the SR family, is encoded by a functional retropseudogene.

scholarly journals Splicing of a Critical Exon of Human Survival Motor Neuron Is Regulated by a Unique Silencer Element Located in the Last Intron

2006 ◽  
Vol 26 (4) ◽  
pp. 1333-1346 ◽  
Author(s):  
Nirmal K. Singh ◽  
Natalia N. Singh ◽  
Elliot J. Androphy ◽  
Ravindra N. Singh
Keyword(s):  
Motor Neuron ◽  
Antisense Oligonucleotide ◽  
Muscular Atrophy ◽  
Dominant Role ◽  
Survival Motor Neuron ◽  
Exon Junction Complex ◽  
Splicing Silencer ◽  
Smn Protein ◽  
First Time

ABSTRACT Humans have two nearly identical copies of the Survival Motor Neuron (SMN) gene, SMN1 and SMN2. In spinal muscular atrophy (SMA), SMN2 is not able to compensate for the loss of SMN1 due to exclusion of exon 7. Here we describe a novel inhibitory element located immediately downstream of the 5′ splice site in intron 7. We call this element intronic splicing silencer N1 (ISS-N1). Deletion of ISS-N1 promoted exon 7 inclusion in mRNAs derived from the SMN2 minigene. Underlining the dominant role of ISS-N1 in exon 7 skipping, abrogation of a number of positive cis elements was tolerated when ISS-N1 was deleted. Confirming the silencer function of ISS-N1, an antisense oligonucleotide against ISS-N1 restored exon 7 inclusion in mRNAs derived from the SMN2 minigene or from endogenous SMN2. Consistently, this oligonucleotide increased the levels of SMN protein in SMA patient-derived cells that carry only the SMN2 gene. Our findings underscore for the first time the profound impact of an evolutionarily nonconserved intronic element on SMN2 exon 7 splicing. Considering that oligonucleotides annealing to intronic sequences do not interfere with exon-junction complex formation or mRNA transport and translation, ISS-N1 provides a very specific and efficient therapeutic target for antisense oligonucleotide-mediated correction of SMN2 splicing in SMA.

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