Arabidopsis SME interactome reveals spliceosome components
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap
ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)
TISSUE(S): Cell Suspension Culture
SUBMITTER: 
Patrick Willems
LAB HEAD: Frank Van Breusegem
PROVIDER: PXD016607 | Pride | 2023-03-03
REPOSITORIES: Pride
Similar Datasets
Project description:Pre-mRNA splicing catalyzed by the spliceosome represents a critical step in the regulation of gene expression contributing to transcriptome and proteome diversity. The spliceosome consists of five small nuclear ribonucleoprotein particles (snRNPs) biogenesis of which remains only partially understood. Here we define the evolutionarily conserved protein Ecdysoneless (Ecd) as a critical regulator of U5 snRNP assembly and Prp8 stability. Combining Drosophila genetics with proteomic approaches we demonstrate Ecd requirement for the maintenance of adult healthspan and lifespan and identify the Sm ring protein SmD3 as a novel interaction partner of Ecd. We show that the predominant task of Ecd is to deliver Prp8 to the Sm protein ring within emerging U5 snRNPs. Ecd deficiency leads to reduced Prp8 protein levels and compromised U5 snRNP biogenesis, causing loss of splicing fidelity and transcriptome integrity. Based on our findings, we propose that Ecd acts as a chaperone that delivers Prp8 to the forming U5 snRNP allowing completion of the cytoplasmic part of the U5 snRNP biogenesis necessary to meet the demand of cells for functional spliceosomes.
2021-04-21 | GSE149056 | GEO
Project description:Background: Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. Results: We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. Conclusions: This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism. RNA-Immunoprecipitation sequencing of RNA-Sm protein complexes.
2013-12-01 | E-GEOD-35842 | biostudies-arrayexpress
Project description:Background: Sm proteins are multimeric RNA-binding factors, found in all three domains of life. Eukaryotic Sm proteins, together with their associated RNAs, form small ribonucleoprotein (RNP) complexes important in multiple aspects of gene regulation. Comprehensive knowledge of the RNA components of Sm RNPs is critical for understanding their functions. Results: We developed a multi-targeting RNA-immunoprecipitation sequencing (RIP-seq) strategy to reliably identify Sm-associated RNAs from Drosophila ovaries and cultured human cells. Using this method, we discovered three major categories of Sm-associated transcripts: small nuclear (sn)RNAs, small Cajal body (sca)RNAs and mRNAs. Additional RIP-PCR analysis showed both ubiquitous and tissue-specific interactions. We provide evidence that the mRNA-Sm interactions are mediated by snRNPs, and that one of the mechanisms of interaction is via base pairing. Moreover, the Sm-associated mRNAs are mature, indicating a splicing-independent function for Sm RNPs. Conclusions: This study represents the first comprehensive analysis of eukaryotic Sm-containing RNPs, and provides a basis for additional functional analyses of Sm proteins and their associated snRNPs outside of the context of pre-mRNA splicing. Our findings expand the repertoire of eukaryotic Sm-containing RNPs and suggest new functions for snRNPs in mRNA metabolism.
2013-12-01 | GSE35842 | GEO
Project description:Alternative splicing is an important posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, termed the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Sm protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase-deficient plants. Furthermore, sme1-2 mutants showed increased tolerance to the superoxide-generating herbicide methyl viologen. mRNA-seq and proteomic analyses indicated a constitutive stress response and pre-mRNA splicing alterations in sme1-2 mutants under control conditions. With a pulldown experiment, using SME1 as a bait, we provide experimental evidence for proteins associated with Arabidopsis thaliana spliceosome complexes, by identifying almost 50 proteins homologous to mammalian spliceosome-associated proteins and propose four novel proteins previously not-reported or hypothesized to be part of plant spliceosome complexes. Mutation of one of these interactors, the Sm core assembly protein ICLN also resulted in a decreased sensitivity to methyl viologen. Taken together, we show that SME1 mutation and impaired snRNP assembly result in enhanced resilience to oxidative stress.
2023-03-03 | PXD024816 | Pride
Project description:Adaptor protein (AP) complexes are evolutionarily conserved vesicle transport regulators that recruit coat proteins, membrane cargos and coated vesicle accessory proteins. Since in plants endocytic and post-Golgi trafficking intersect at the trans-Golgi network, unique mechanisms for sorting cargos of overlapping vesicular routes are anticipated. The plant AP complexes are part of the sorting machinery, but despite some functional information, their cargoes, accessory proteins, and regulation remain largely unknown. Here, by means of various proteomics approaches, we generated the overall interactome of the five AP and the TPLATE complexes in Arabidopsis thaliana. The interactome converged on a number of hub proteins, including the thus far unknown adaptin binding-like protein, designated P34. P34 interacted with the clathrin-associated AP complexes, controlled their stability and, subsequently, influenced clathrin-mediated endocytosis and various post-Golgi trafficking routes. Altogether, the AP interactome network offers substantial resources for further discoveries of unknown endomembrane trafficking regulators in plant cells.
2023-12-07 | PXD035454 | Pride
Project description:Spliceosomal snRNA are key components of small nuclear ribonucleoprotein particles (snRNPs), the building blocks of the spliceosome. The biogenesis of snRNPs is a complex process involving multiple cellular and subcellular compartments, the details of which are yet to be described. In short, the snRNA is exported to the cytoplasm as 3‘-end extended precursor (pre-snRNA), where it acquires a heptameric Sm ring. The SMN complex which catalyses this step, recruits Sm proteins and assembles them around the pre-snRNA at the single stranded Sm site. After additional modification, the complex is re-imported into the nucleus where the final maturation step occurs. Our modeling suggests that during the cytoplasmic stage of maturation pre-snRNA assumes a compact secondary structure containing Near Sm site Stem (NSS) which is not compattible with the formation of the Sm ring. To validate our in silico predictions we employed selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) on U2 snRNA in vivo, ex vivo and in vitro, and U4 pre-snRNA in vitro. For the in vivo experiment HeLa cells were incubated for 10 min at 37°C with NAI or DMSO to final concentration 200 mM. RNA was isolated using Trizol (Sigma) and 200 µl chloroform and precipitated with ethanol at -20°C overnight. For the ex vivo experiment, RNA was isolated from HeLa cells after Protease K treatment at room temperature for 45 min. After incubation, RNA was isolated using equilibrated phenol/chloroform/isoamyl alcohol buffered by folding buffer (110 mM HEPES pH 8.0, 110 mM KCl, 11 mM MgCl2) and cleaned on a PD-10 column according to the manufacturer’s instructions. Isolated RNA was treated with 100mM NAI or DMSO for 10 min at 37°C. For the in vitro experiment, U2WT and U4 pre-snRNA were transcribed by T7 polymerase followed by DNase I (30 min at 37 °C) and Proteinase K (30 min at 37°C) treatments. U2 snRNA was purified on 30 kDa Amicon columns, folded for 30 min at 37°C in 57 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. U4 pre-snRNA was purified on Superdex 200 Increase 10/300GL, folded for 30 min at 37°C in 60 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. All prepared RNA samples (in vitro, ex vivo, in vivo) were used for reverse transcription with the gene-specific primer 5’-CGTTCCTGGAGGTACTGCAA for U2 snRNA and 5’- AAAAATTCAGTCTCCG for U4 pre-snRNA. We used SHAPE MaP buffer (50 mM Tris-HCl pH 8.0, 75 mM KCl, 10 mM DTT, 0.5 mM dNTP, 6 mM MnCl2) and SuperScript II (Invitrogen). Amplicons for snRNAs were generated using gene-specific forward and reverse primers. Importantly, the primers include Nextera adaptors required for downstream library construction. PCR reaction products were cleaned using Monarch PCR&DNA Clean-up Kits. Remaining Illumina adaptor sequences were added using the PCR MasterMix and index primers provided in the NexteraXT DNA Library Preparation Kit (Illumina) according to the manufacturer’s protocol. Libraries were quantified using Qubit (Invitrogen) and BioAnalyzer (Agilent). Amplicons were sequenced on a NextSeq 500/550 platform using a 150 cycle mid-output kit. All sequencing data was analyzed using the ShapeMapper 2 analysis pipeline1. The ‘—amplicon’ and ‘—primers’ flags were used, along with sequences of gene-specific handles PCR primers, to ensure primer binding sites are excluded from reactivity calculations. Default read-depth thresholds of 5000x were used. Analysis of statistically significant reactivity differences between ex vivo and in vivo-determined SHAPE reactivities was performed using the DeltaSHAPE automated analysis tool and default settings2. 1. Busan, S. & Weeks, K.M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24, 143-148 (2018). 2. Smola, M.J., Rice, G.M., Busan, S., Siegfried, N.A. & Weeks, K.M. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10, 1643-69 (2015).
2023-09-08 | E-MTAB-13248 | biostudies-arrayexpress
Project description:Salt stress caused by soil salination inhibits plant growth and development that result in reduction of crop yield and threaten the food security. Several spliceosome components are considered to modify salt stress responses in plants. However, the molecular basis of spliceosome proteins adjustment to salt stress is still unclear. Here we report that an Sm core protein SmEb is required for salt tolerance in Arabidopsis. In addition, SmEb controls alternative splicing of hundreds of pre-mRNA to participate in plant response to salt stress. Our results further reveal that SmEb takes effect on maintain proper ratio of two RCD1 splicing variants to adjust to H2O2 accumulation under salt stress. Together, our findings uncover that proper alternative splicing of pre-mRNAs governed by the spliceosome component SmEb is essential for plant salt stress responses. Salt stress caused by soil salination inhibits plant growth and development that result in reduction of crop yield and threaten the food security. Several spliceosome components are considered to modify salt stress responses in plants. However, the molecular basis of spliceosome proteins adjustment to salt stress is still unclear. Here we report that an Sm core protein SmEb is required for salt tolerance in Arabidopsis. In addition, SmEb controls alternative splicing of hundreds of pre-mRNA to participate in plant response to salt stress. Our results further reveal that SmEb takes effect on maintain proper ratio of two RCD1 splicing variants to adjust to H2O2 accumulation under salt stress. Together, our findings uncover that proper alternative splicing of pre-mRNAs governed by the spliceosome component SmEb is essential for plant salt stress responses.
2023-04-01 | GSE208786 | GEO
Project description:U4/U6 di-snRNPs were disrupted and singular U4 and U6 snRNPs accumulated in egy mutant embryos, establishing the recycling function of p110 in vivo. Based on microarray analyses, a subset of spliceosome components and splicing-related factors was coordinately upregulated in the egy mutant. This revealed an extensive network of coregulated components of the spliceosome cycle, compensating – albeit inefficiently – for the recycling defect. In contrast, another set of genes, many of them eye- and pancreas-specific, was downregulated in the egy mutant embryos. Keywords: mut / wt comparison
2007-01-31 | GSE5586 | GEO
Project description:Various genetic diseases associated with microcephaly and developmental defects are due to pathogenic variants in the U4atac snRNA, a component of the minor spliceosome essential for the removal of U12-type introns from eukaryotic messenger RNAs. While it has been shown that a few RNU4ATAC mutations result in impaired binding of essential protein components, the molecular defects of the vast majority are still unknown. Here, we used cells derived from two RNU4ATAC compound heterozygous (g.108_126del;g.111G>A) twin patients with MOPD1 phenotypes to analyze the molecular consequences of the mutations on snRNPs formation and splicing. We show that the U4atac108_126del mutant is unstable and that the 111G>A mutation located in the apical 3’ stem-loop of U4atac induces defects in 3’-end maturation giving rise to decreased levels of mature U4atacG111A snRNA as well as of minor di- and tri-snRNPs. We also found that the mutant cells have alterations in splicing of minor introns and contain lower levels of INTS7 and INTS10 proteins. Altogether, our results demonstrate that the compound heterozygous g.108_126del;g.111G>A U4atac mutations lead to defects in 3’-end processing of U4atac transcripts and affect the homeostasis and function of the Integrator complex.
2022-12-01 | GSE197344 | GEO
Project description:Circadian clocks generate endogenous rhythms in most organisms from cyanobacteria to humans and facilitate entrainment to environmental diurnal cycles, thus conferring a fitness advantage. Both transcriptional and posttranslational mechanisms are prominent in the basic network architecture of circadian systems. Posttranscriptional regulation, including mRNA processing, is emerging as a critical step for the clock function. However, little is known about the molecular mechanisms linking RNA metabolism to the circadian clock network. Here we report that a conserved SNW/SKIP domain protein, SKIP, a splicing factor and component of the spliceosome, is involved in the posttranscriptional regulation of circadian clock genes in Arabidopsis. Mutation in SKIP lengthens the circadian period in a temperature sensitive manner, affects light input and the sensitivity of light resetting to the clock. SKIP physically interacts with the spliceosomal splicing factor SR45 and associates with the pre-mRNA of clock genes, such as PRR7 and PRR9, and is necessary for the regulation of their alternative splicing and mRNA maturation. Genome-wide investigations reveal that SKIP functions in regulating alternative splicing of many genes, presumably through modulating recognition or cleavage of 5' and 3' splicing site. Our study addresses a fundamental question on how the mRNA splicing machinery contributes to circadian clock functions at a posttranscriptional level. Our findings revealed that AtSKIP is a splicing factor and a component of spliceosome. To further investigate effects of mutation in SKIP on the genome-wide changes of alternative splicing, we performed ultra-highthroughput RNA sequencing, using 10-day old seedlings. Two biological replicates for both wild type (Col-0) and skip-2 were designed.
2012-12-13 | E-GEOD-32216 | biostudies-arrayexpress