scholarly journals Definition of a RACK1 Interaction Network in Drosophila melanogaster Using SWATH-MS

2017 ◽  
Vol 7 (7) ◽  
pp. 2249-2258 ◽  
Author(s):  
Lauriane Kuhn ◽  
Karim Majzoub ◽  
Evelyne Einhorn ◽  
Johana Chicher ◽  
Julien Pompon ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Activated Protein C ◽  
Model Organism ◽  
Interaction Network ◽  
Added Value ◽  
Label Free ◽  
Nucleic Acid Binding ◽  
Entry Site

Abstract Receptor for Activated protein C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism Drosophila melanogaster, we recently showed that RACK1 is required at the ribosome for internal ribosome entry site (IRES)-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in Drosophila S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition (DDA and DIA, respectively) and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method, both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for Drosophila and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation, and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus (CrPV), an IRES-containing virus.

scholarly journals A unique ribonucleoprotein complex assembles preferentially on ecdysone-responsive sites in Drosophila melanogaster.

1993 ◽  
Vol 13 (9) ◽  
pp. 5323-5330 ◽  
Author(s):  
S A Amero ◽  
M J Matunis ◽  
E L Matunis ◽  
J W Hockensmith ◽  
G Raychaudhuri ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Polytene Chromosomes ◽  
Cell Extract ◽  
Sequence Motifs ◽  
Drosophila Cell ◽  
Rnase Digestion

The protein on ecdysone puffs (PEP) is associated preferentially with active ecdysone-inducible puffs on Drosophila polytene chromosomes and contains sequence motifs characteristic of transcription factors and RNA-binding proteins (S. A. Amero, S. C. R. Elgin, and A. L. Beyer, Genes Dev. 5:188-200, 1991). PEP is associated with RNA in vivo, as demonstrated here by the sensitivity of PEP-specific chromosomal immunostaining in situ to RNase digestion and by the immunopurification of PEP in Drosophila cell extract with heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As revealed by sequential immunostaining, PEP is found on a subset of chromosomal sites bound by the HRB (heterogeneous nuclear RNA-binding) proteins, which are basic Drosophila hnRNPs. These observations lead us to suggest that a unique, PEP-containing hnRNP complex assembles preferentially on the transcripts of ecdysone-regulated genes in Drosophila melanogaster presumably to expedite the transcription and/or processing of these transcripts.

scholarly journals Premature Translation of oskar in Oocytes Lacking the RNA-Binding Protein Bicaudal-C

1998 ◽  
Vol 18 (8) ◽  
pp. 4855-4862 ◽  
Author(s):  
Emma E. Saffman ◽  
Sylvia Styhler ◽  
Katherine Rother ◽  
Weihua Li ◽  
Stéphane Richard ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Point Mutation ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Binding Protein ◽  
C Protein ◽  
Kh Domain ◽  
Anterior Posterior

ABSTRACT Bicaudal-C (Bic-C) is required duringDrosophila melanogaster oogenesis for several processes, including anterior-posterior patterning. The gene encodes a protein with five copies of the KH domain, a motif found in a number of RNA-binding proteins. Using antibodies raised against the BIC-C protein, we show that multiple isoforms of the protein exist in ovaries and that the protein, like the RNA, accumulates in the developing oocyte early in oogenesis. BIC-C protein expressed in mammalian cells can bind RNA in vitro, and a point mutation in one of the KH domains that causes a strong Bic-C phenotype weakens this binding. In addition, oskar translation commences prior to posterior localization of oskar RNA inBic-C − oocytes, indicating thatBic-C may regulate oskar translation during oogenesis.

scholarly journals RNA regulons and the RNA-protein interaction network

2012 ◽  
Vol 3 (5) ◽  
pp. 403-414 ◽  
Author(s):  
Jochen Imig ◽  
Alexander Kanitz ◽  
André P. Gerber
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Interaction Network ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Interaction Network ◽  
Coordinated Control ◽  
Non Coding Rnas ◽  
Rna Interaction

AbstractThe development of genome-wide analysis tools has prompted global investigation of the gene expression program, revealing highly coordinated control mechanisms that ensure proper spatiotemporal activity of a cell’s macromolecular components. With respect to the regulation of RNA transcripts, the concept of RNA regulons, which – by analogy with DNA regulons in bacteria – refers to the coordinated control of functionally related RNA molecules, has emerged as a unifying theory that describes the logic of regulatory RNA-protein interactions in eukaryotes. Hundreds of RNA-binding proteins and small non-coding RNAs, such as microRNAs, bind to distinct elements in target RNAs, thereby exerting specific and concerted control over posttranscriptional events. In this review, we discuss recent reports committed to systematically explore the RNA-protein interaction network and outline some of the principles and recurring features of RNA regulons: the coordination of functionally related mRNAs through RNA-binding proteins or non-coding RNAs, the modular structure of its components, and the dynamic rewiring of RNA-protein interactions upon exposure to internal or external stimuli. We also summarize evidence for robust combinatorial control of mRNAs, which could determine the ultimate fate of each mRNA molecule in a cell. Finally, the compilation and integration of global protein-RNA interaction data has yielded first insights into network structures and provided the hypothesis that RNA regulons may, in part, constitute noise ‘buffers’ to handle stochasticity in cellular transcription.

scholarly journals RNA-binding Protein HuR Interacts with Thrombomodulin 5′Untranslated Region and Represses Internal Ribosome Entry Site–mediated Translation under IL-1β Treatment

2008 ◽  
Vol 19 (9) ◽  
pp. 3812-3822 ◽  
Author(s):  
Chiu-Hung Yeh ◽  
Liang-Yi Hung ◽  
Chin Hsu ◽  
Shu-Yun Le ◽  
Pin-Tse Lee ◽  
...  
Keyword(s):  
Protein Expression ◽  
Internal Ribosome Entry Site ◽  
Untranslated Region ◽  
Protein C ◽  
Rna Binding ◽  
Activated Protein C ◽  
Critical Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Sepsis And Septic Shock

Reduction in host-activated protein C levels and resultant microvascular thrombosis highlight the important functional role of protein C anticoagulant system in the pathogenesis of sepsis and septic shock. Thrombomodulin (TM) is a critical factor to activate protein C in mediating the anticoagulation and anti-inflammation effects. However, TM protein content is decreased in inflammation and sepsis, and the mechanism is still not well defined. In this report, we identified that the TM 5′ untranslated region (UTR) bearing the internal ribosome entry site (IRES) element controls TM protein expression. Using RNA probe pulldown assay, HuR was demonstrated to interact with the TM 5′UTR. Overexpression of HuR protein inhibited the activity of TM IRES, whereas on the other hand, reducing the HuR protein level reversed this effect. When cells were treated with IL-1β, the IRES activity was suppressed and accompanied by an increased interaction between HuR and TM 5′UTR. In the animal model of sepsis, we found the TM protein expression level to be decreased while concurrently observing the increased interaction between HuR and TM mRNA in liver tissue. In summary, HuR plays an important role in suppression of TM protein synthesis in IL-1β treatment and sepsis.

scholarly journals Matrin 3-dependent neurotoxicity is modified by nucleic acid binding and nucleocytoplasmic localization

2018 ◽  
Author(s):  
Ahmed M. Malik ◽  
Roberto A. Miguez ◽  
Xingli Li ◽  
Ye-Shih Ho ◽  
Eva L. Feldman ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Self Assembly ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleic Acid Binding ◽  
Rna Recognition Motifs ◽  
Matrin 3 ◽  
Dna And Rna ◽  
Acid Processing ◽  
Recognition Motifs

ABSTRACTAbnormalities in nucleic acid processing are associated with the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in Matrin 3 (MATR3), a poorly understood DNA- and RNA-binding protein, cause familial ALS/FTD, and MATR3 pathology is a feature of sporadic disease, suggesting that MATR3 dysfunction is integrally linked to ALS pathogenesis. Using a primary neuron model to assess MATR3-mediated toxicity, we noted that neurons were bidirectionally vulnerable to MATR3 levels, with pathogenic MATR3 mutants displaying enhanced toxicity. MATR3’s zinc finger domains partially modulated toxicity, but elimination of its RNA recognition motifs had no effect on neuronal survival, instead facilitating its self-assembly into liquid-like droplets. In contrast to other RNA-binding proteins associated with ALS, cytoplasmic MATR3 redistribution mitigated neurodegeneration, suggesting that nuclear MATR3 mediates toxicity. Our findings offer a foundation for understanding MATR3-related neurodegeneration and how nucleic acid binding functions, localization, and pathogenic mutations drive sporadic and familial disease.

scholarly journals Hnrnpul1 loss of function affects skeletal and limb development

2020 ◽  
Author(s):  
Danielle L Blackwell ◽  
Sherri D Fraser ◽  
Oana Caluseriu ◽  
Claudia Vivori ◽  
Paul MK Gordon ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Limb Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleic Acid Binding ◽  
Loss Of Function ◽  
Zebrafish Model ◽  
Dna And Rna ◽  
Developmental Role

AbstractMutations in RNA binding proteins can lead to pleiotropic phenotypes including craniofacial, skeletal, limb and neurological symptoms. Heterogeneous Nuclear Ribonucleoproteins (hnRNPs) are involved in nucleic acid binding, transcription and splicing through direct binding to DNA and RNA, or through interaction with other proteins in the spliceosome. Here, we show a developmental role for hnrnpul1 in zebrafish fin and craniofacial development, and in adult onset scoliosis. Furthermore, we demonstrate a role of hnrnpul1 in alternative splicing regulation. In two siblings with congenital limb malformations, whole exome sequencing detected a frameshift variant in HNRNPUL1; the developmental role of this gene in humans has not been explored. Our data suggest an important developmental role of hnRNPUL1 in both zebrafish and humans. Although there are differences in phenotypes between species, our data suggests potential conservation of ancient regulatory circuits involving hnRNPUL1 in these phylogenetically distant species.Summary statementA zebrafish model of loss of Hnrnpul1 shows alternative splicing defects and results in limb growth, craniofacial tendon, and skeletal anomalies.

scholarly journals An interaction network of RNA-binding proteins involved in Drosophila oogenesis

2020 ◽  
Author(s):  
Prashali Bansal ◽  
Johannes Madlung ◽  
Kristina Schaaf ◽  
Boris Macek ◽  
Fulvia Bono
Keyword(s):  
Translational Regulation ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Direct Interaction ◽  
Interaction Network ◽  
Splicing Factors ◽  
Mrna Regulation ◽  
Interaction Partners ◽  
Maternal Transcripts

AbstractDuring Drosophila oogenesis, the localization and translational regulation of maternal transcripts relies on RNA-binding proteins (RBPs). Many of these RBPs localize several mRNAs and may have additional direct interaction partners to regulate their functions. Using immunoprecipitation from whole Drosophila ovaries coupled to mass spectrometry, we examined protein-protein associations of 6 GFP-tagged RBPs expressed at physiological levels. Analysis of the interaction network and further validation in human cells allowed us to identify 26 previously unknown associations, besides recovering several well characterized interactions. We identified interactions between RBPs and several splicing factors, providing links between nuclear and cytoplasmic events of mRNA regulation. Additionally, components of the translational and RNA decay machineries were selectively co-purified with some baits, suggesting a mechanism for how RBPs may regulate maternal transcripts. Given the evolutionary conservation of the studied RBPs, the interaction network presented here provides the foundation for future functional and structural studies of mRNA localization across metazoans.

scholarly journals Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins

1987 ◽  
Vol 7 (8) ◽  
pp. 2947-2955
Author(s):  
A Y Jong ◽  
M W Clark ◽  
M Gilbert ◽  
A Oehm ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Nucleic Acid ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleic Acid Binding ◽  
Dna And Rna ◽  
C Terminus

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Multiple Roles of RNA Regulatory Factors in Neuronal Development and Function in C. elegans

2020 ◽  
Author(s):  
Matthew G. Andrusiak ◽  
Yishi Jin
Keyword(s):  
Nervous System ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Model Organism ◽  
Nervous System Development ◽  
Forward Genetics ◽  
C Elegans

Recent evidence has highlighted the dynamic nature of mRNA regulation, particularly in the nervous system, from complex pre-mRNA processing to long-range transport and long-term storage of mature mRNAs. In accordance with the importance for mRNA-mediated regulation of nervous system development and maintenance, various mutations in RNA-binding proteins are associated with a range of human disorders. C. elegans express many RNA-binding factors that have human orthologs and perform similar biochemical functions. This chapter focuses on the research using C. elegans to dissect molecular mechanisms involving mRNA-mediated pathways. It highlights the key approaches and findings that integrate genetic and genomic studies in the nervous system. The analyses of genetic mutants, primarily using forward genetics, offer functional insights for genes important for neuronal development, synaptic transmission, and neuronal repair. In combination with single-neuron cell biology and cell-type genomics, the knowledge learned from this model organism has continued to lead to ground-breaking discoveries.

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