Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA97849 | ENA |
REPOSITORIES: ENA
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Project description:Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well. Keywords: time course, splicing mutant, splicing-specific microarray
2007-04-04 | GSE7419 | GEO
Project description:Pre-mRNA splicing is a precise regulated process, and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs), and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remain largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In this study, we generated a prpf31 knockout zebrafish line, and discovered that prpf31 mutants exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31 deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.
2022-04-07 | GSE171661 | GEO
Project description:The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of AdML – a model pre-mRNA substrate – guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5′ and 3′ splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a three-dimensional structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable three-dimensional structural scaffolds to the splicing code across the mammalian transcriptome.
2021-06-23 | GSE173175 | GEO
Project description:The specific recognition of splice signals at or near the exon-intron junctions is not explained by their weak conservation across the mammalian transcriptome and postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of a pre-mRNA guiding early spliceosomal components to the splice signal sequences. We find that mutation in non-cognate splice signal sequences of a model pre-mRNA substrate could impede recruitment of early spliceosomal components due to disruption of global structure of the pre-mRNA. We also find distribution of pre-mRNA segments potentially interacting with early spliceosomal component U1 snRNP across the intron, spatial proximity of 5′ and 3′ splice sites within the pre-mRNA scaffold, and an interplay between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components could recognize a three-dimensional structural scaffold beyond the short splice signal sequences and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This work provides a conceptual base to extend our understanding of prevalence, distribution, and splicing regulatory potential of recognizable three-dimensional structural scaffolds across the mammalian transcriptome.
2021-06-23 | GSE173177 | GEO
Project description:RNA splicing and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice we show that XAB2, the human homologue of the yeast pre-mRNA-splicing factor SYF1 has a functional role in Nucleotide Excision Repair (NER) and the DNA damage response (DDR) in mammals. XAB2 interacts with spliceosome factors and is part of the core spliceosome that binds to spliceosomal U4 and U6 snRNAs during hepatic development. Ablation of XAB2 leads to defective NER, intron retention, the aberrant accumulation of pre-mRNAs and to a faulty ATM/ATR DDR signaling. Using functional approaches, we find that XAB2 dissociates from RNA targets upon persistent DNA damage or transcription blockage and from spliceosomal RNAs in the NER-defective developing livers. Thus, XAB2 functionally links NER to the spliceosomal response to DNA damage during hepatic development with important ramifications for transcription-coupled DNA repair disorders.
2021-09-08 | PXD014084 | Pride
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
Project description:We determined that over 60 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G-patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.
2021-03-13 | GSE168814 | GEO
Project description:The spliceosome is a dynamic RNA-protein complex that executes pre-mRNA splicing and is composed of five core small nuclear ribonucleoprotein particles (U1, U2, U4/5/6 snRNP) and >150 additional proteins specific for each snRNP. We report a circadian role for Pre-mRNA Processing factor 4 (PRP4), a conserved component of the spliceosomal U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex. We broadly hypothesized that downregulation of prp4 led to the aberrant splicing of one or many of the core clock transcripts. To identify these splicing events in an unbiased way, we performed RNA-Sequencing (RNA-Seq) analysis. We reasoned that we could have a more targeted approach if we could zoom in on the overlapping splicing changes that would be driven by the knockdown of at least two different tri-snRNP components. Because the pan-neuronal knockdown of all tri-snRNP components tested in our study led to lethality, we decided to utilize an alternative broad driver. For that purpose, we selected a strong eye-specific Glass Multiple Promoter driver (GMR-Gal4). Because most of the signal from head lysates comes directly from the eye tissue and because the core splicing factors are ubiquitously expressed, GMR-specific downregulation of prp4 and prp8 promised to be a viable alternative to the pan-neuronal knockdown. We examined changes in both the total transcript levels and splicing events upon prp4 knockdown in the eye. The overall gene expression seemed to be dramatically influenced by prp4 downregulation (433 DOWN, 310 UP at FDR < 0.05). Despite the fact that PRP4 is a component of the core spliceosome that is required for constitutive exon splicing, we did not detect dramatic effects on global splicing. Only 45 genes exhibited differential alternate splicing upon prp4 downregulation at FDR < 0.05).
2018-11-30 | GSE115163 | GEO
Project description:Pre-mRNA splicing relies on the still poorly understood dynamic interplay between more than 150 protein components of the Spliceosome, and the steps at which splicing can be regulated remain largely unknown. Here we systematically analyze the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and use this information to reconstruct a network of functional interactions. The network accurately captures well-established physical and functional associations and identifies new, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during Spliceosome assembly. In contrast with standard models of regulation at early events of splice site recognition, factors involved in catalytic activation of the Spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF and on targets of anti-tumor drugs, and can be widely used to identify mechanisms of splicing regulation. RNA from 3 biological replicates of 72 hours knockdowns of human IK or SMU1 and a control set were used. Changes between the control and knockdowns were measured based on using a splice-junction array (Affymetrix HJAY).
2015-12-31 | E-GEOD-56605 | biostudies-arrayexpress
Project description:Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome––the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal subcomplexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and USH. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.
2021-05-25 | PXD023942 | Pride