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Differential pre-mRNA defects in fission yeast carrying a temperature degron allele of the SMN gene

ABSTRACT: The SMA disease results from deletions or mutations in the “survival of motor neuron” (SMN1) gene. The SMN protein plays an essential role in the biogenesis of spliceosomal snRNPs but the link between a defect in this process and specific splicing inhibition is not known. Here, we report the construction of a temperature degron (td) allele of the S. pombe SMN protein and show that its depletion at 37°C affects snRNPs stability and splicing. In addition, the function of the tdSMN allele in snRNPs assembly is affected already at 25°C suggesting a deleterious effect of the tag at this temperature. Remarkably, using S. pombe tiling arrays and RT-PCR validation tests, we report that splicing is altered differentially in the tdSMN cells at permissive temperature. Our results suggest that the defects observed in tdSMN fission yeast cells mimic splicing deficits observed in SMN-deficient metazoan cells.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE17010 | GEO | 2010/04/22

SECONDARY ACCESSION(S): PRJNA117751

REPOSITORIES: GEO

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Trancriptional regulation by axonal SMN on motor neuron cells

Project description:Muscular atrophy (SMA) is an autosomal recessive disease causing selective motor neuron death by the loss of telomeric survival motor neuron gene, SMN1. Axonal SMN, a-SMN, is a truncated form of SMN, derived from an alternatively spliced SMN1 gene. (Setola, et. al. 2007 PNAS 104, 1959-1964). The cellular clones expressing a-SMN in a tetracycline-dependent manner were isolated from NSC34 by two-step stable transfection, first with the tetracycline-repressor construct and subsequently with the a-SMN cDNA. To identify novel a-SMN target genes, the transcriptome of several a-SMN clones was analyzed and compared with that of parental cells.

2012-01-07 | E-MEXP-3402 | biostudies-arrayexpress

Mass spectrometry-based proteomic comparison of BirA-SMN, BirA-SMN-Y109C and BirA proxisomes.

Project description:Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology that originates from the loss of the SMN1 gene in most cases or mutations in rare cases. Interestingly, SMN1 mutations occur broadly either within the TUDOR methyl-arginine (Rme) reader domain of SMN or its carboxy terminal oligomerization domain. We hypothesized that in SMN1 mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a proximity-dependent biotinylation proteomic (BioID) approach, we have identified and validated a number of SMN-interacting proteins, including Fragile X mental retardation family members (FMRFM), such as FMRP itself as well as FXR1 and FXR2, some depending on a wild-type version of the TUDOR domain.

2022-10-19 | PXD030970 | Pride

Gain of toxic function by long-term SMN overexpression in the sensory-motor circuit.

Project description:The neurodegenerative disease spinal muscular atrophy (SMA) is a leading genetic cause of infant death caused by deficiency in the survival motor neuron (SMN) protein. Currently approved SMA treatments aim to restore SMN, but the potential for expression of SMN beyond physiological levels is a unique feature of AAV9-SMN gene therapy. Here, we show that long-term AAV9-mediated SMN overexpression in mouse models induces dose-dependent, late-onset motor dysfunction associated with loss of proprioceptive synapses and neurodegeneration. Mechanistically, aggregation of overexpressed SMN in the cytoplasm of motor circuit neurons sequesters components of small nuclear ribonucleoproteins (snRNPs), leading to splicing dysregulation and widespread transcriptome abnormalities with prominent signatures of neuroinflammation and innate immune response. Thus, long-term SMN overexpression can interfere with its normal activity in RNA regulation and trigger SMA-like pathogenic events through toxic gain of function mechanisms. These unanticipated, SMN-dependent and neuron-specific liabilities of AAV9-SMN warrant further evaluation of the long-term safety of gene therapy in SMA.

2021-02-01 | GSE149391 | GEO

Interaction of 7SK with the Smn complex modulates snRNP production

Project description:Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.

2021-01-26 | PXD021360 | Pride

SMN deficiency in spinal muscular atrophy causes widespread intron retention and DNA damage

Project description:Spinal muscular atrophy is the leading genetic cause of infant mortality and is caused by homozygous loss of the SMN1 gene. We investigated global transcriptome changes in the spinal cord of inducible SMA mice. SMN depletion caused widespread retention of introns with weak splice sites or belonging to the minor (U12) class. We further demonstrated accumulation of DNA double strand breaks in the spinal cord of SMA mice and in human SMA cell culture models. DNA damage was partially rescued by suppressing the formation of R-loops, which accumulated over retained introns. We propose that instead of single gene effects, pervasive splicing defects caused by SMN deficiency trigger a global DNA damage and stress response, thus compromising motor neuron survival.

2017-03-21 | GSE87281 | GEO

Characterization and in vivo functional analysis of the Schizosaccharomyces pombe ICln gene

Project description:During the early steps of snRNP biogenesis, the Survival of Motor Neuron (SMN) complex acts together with the methylosome, an entity formed by the pICln protein, WD45 and the PRMT5 methyltransferase. To expand our understanding of pICln and SMN functional relationships in vivo, we performed a genetic analysis of an uncharacterized S. pombe pICLn homologue. Although not essential, the S. pombe ICln protein is important for optimal yeast cell growth. The human pICln gene complements the iclnM-bM-^HM-^F slow growth phenotype demonstrating that the identified SpICln sequence represents the bona fide human homolog. Consistent with the role inferred for human pICln using in vitro experiments, we found that the SpICln protein is required for optimal production of the spliceosomal snRNPs and for efficient splicing in vivo. Genetic interaction approaches demonstrate furthermore that modulation of ICln activity is unable to compensate for defects induced by SMN mutations, and reciprocally. Using a genome-wide approach and RT-PCR validation tests, we show also that splicing is altered differentially in iclnM-bM-^HM-^F cells. Our data are consistent with the emerging view that splice site selection and spliceosome kinetics is highly dependent on the concentration of core spliceosomal components. RNA from M-NM-^TIcln mutant (2 replicates) vs RNA from wild type cells (2 replicates)

2013-12-05 | E-GEOD-41403 | biostudies-arrayexpress

Characterization and in vivo functional analysis of the Schizosaccharomyces pombe ICln gene

Project description:During the early steps of snRNP biogenesis, the Survival of Motor Neuron (SMN) complex acts together with the methylosome, an entity formed by the pICln protein, WD45 and the PRMT5 methyltransferase. To expand our understanding of pICln and SMN functional relationships in vivo, we performed a genetic analysis of an uncharacterized S. pombe pICLn homologue. Although not essential, the S. pombe ICln protein is important for optimal yeast cell growth. The human pICln gene complements the icln∆ slow growth phenotype demonstrating that the identified SpICln sequence represents the bona fide human homolog. Consistent with the role inferred for human pICln using in vitro experiments, we found that the SpICln protein is required for optimal production of the spliceosomal snRNPs and for efficient splicing in vivo. Genetic interaction approaches demonstrate furthermore that modulation of ICln activity is unable to compensate for defects induced by SMN mutations, and reciprocally. Using a genome-wide approach and RT-PCR validation tests, we show also that splicing is altered differentially in icln∆ cells. Our data are consistent with the emerging view that splice site selection and spliceosome kinetics is highly dependent on the concentration of core spliceosomal components.

2013-12-05 | GSE41403 | GEO

Ribosome profiling of SMN-primed ribosomes and active ribosome profiling from brains of a mouse model of severe SMA

Project description:Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality, with an incidence of around 1 in 6,000-10,000 live births. SMA is caused by low levels of full-length survival of motor neuron protein (SMN). We demonstrate that SMN binds to ribosomes and that this interaction is tissue-dependent. We performed ribosome profiling analyses on lysates from P5 wild-type mouse brains exposed to RNase I, from which we isolated SMN-primed ribosomes by immunoprecipitation. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs which are enriched for translational enhancer sequences in the 5’UTR and rare codons at the beginning of their coding sequence. These SMN-specific mRNAs are associated with neurogenesis, lipid metabolism, ubiquitination, chromatin regulation and translation. Additionally, we analyzed the positioning of active ribosomes using the Active-RiboSeq method based on RiboLace and compared early symptomatic SMA mouse brains to age-matched controls. We found that loss of SMN induces ribosome depletion, especially at the beginning of the coding sequence of SMN-specific mRNAs, leading to impairment of proteins involved in motor neuron function and stability. Keywords: motor neuron disease, spinal muscular atrophy, SMA, SMN, SMN1, survival of motor neuron, SMN-primed ribosome profiling, translation, RiboLace, Ribo-Seq, active translation, ribosome heterogeneity

2020-08-11 | GSE154106 | GEO

ALS-causative mutations in FUS/TLS confer gain- and loss-of-function by altered association with SMN and U1-snRNP

Project description:The RNA-binding protein FUS/TLS, mutation in which is causative of the fatal motor neuron disease ALS, is demonstrated to directly bind to the U1-snRNP and SMN complexes. ALS-causative mutations in FUS/TLS are shown to abnormally enhance their interaction with SMN and reduce interaction with U1-snRNP. Correspondingly, global RNA analysis reveals a mutant-dependent loss of splicing activity, with ALS-linked mutants failing to reverse changes caused by loss of wild-type FUS/TLS. Furthermore, a common FUS/TLS mutant-associated RNA splicing signature is identified in ALS patient fibroblasts. Taken together, our studies establish potentially converging disease mechanisms in ALS and spinal muscular atrophy, with ALS-causative mutants acquiring properties representing both gain (dysregulation of SMN) and loss (reduced RNA processing mediated by U1-snRNP) of function. RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Generation sequencing (RASL-seq) method was used for analyzing alternative splicing changes. Oligonucleotide probes are designed to anneal to the exon-exon junctions. The probe library was assembled to assess 5530 unique alternative splicing events, most of which were exon inclusion or skipping, with a minority for alternative 5’- or 3’- splice sites. The splicing changes were compared among groups of reducing FUS/TLS or SMN levels, or expressing various FUS mutations to determine the loss versus gain of FUS/TLS function on splicing regulation.

2015-02-10 | E-GEOD-64078 | biostudies-arrayexpress

Role of SMN Protein in regulating the transcription and alternative splicing of Pulmonary Vascular Remodeling-Related Genes in HPASMCs

Project description:To explore the effect of SMN protein on alternative splicing of multiple genes in human pulmonary artery smooth muscle cells (HPASMCs), we used shRNA specifically against the SMN to knock down the expression of SMN protein.Human primary pulmonary artery smooth muscle cells (hPASMCs) of donors were obtained from China Center for Type Culture Collection (Wuhan, Hubei, China).

2024-03-15 | GSE261233 | GEO

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