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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

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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.

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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

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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

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