142 research outputs found

    Whole transcriptome analysis reveals non-coding RNA's competing endogenous gene pairs as novel form of motifs in serous ovarian cancer

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    Publisher Copyright: © 2022The non-coding RNA (ncRNA) regulation appears to be associated to the diagnosis and targeted therapy of complex diseases. Motifs of non-coding RNAs and genes in the competing endogenous RNA (ceRNA) network would probably contribute to the accurate prediction of serous ovarian carcinoma (SOC). We conducted a microarray study profiling the whole transcriptomes of eight human SOCs and eight controls and constructed a ceRNA network including mRNAs, long ncRNAs, and circular RNAs (circRNAs). Novel form of motifs (mRNA-ncRNA-mRNA) were identified from the ceRNA network and defined as non-coding RNA's competing endogenous gene pairs (ceGPs), using a proposed method denoised individualized pair analysis of gene expression (deiPAGE). 18 cricRNA's ceGPs (cceGPs) were identified from multiple cohorts and were fused as an indicator (SOC index) for SOC discrimination, which carried a high predictive capacity in independent cohorts. SOC index was negatively correlated with the CD8+/CD4+ ratio in tumour-infiltration, reflecting the migration and growth of tumour cells in ovarian cancer progression. Moreover, most of the RNAs in SOC index were experimentally validated involved in ovarian cancer development. Our results elucidate the discriminative capability of SOC index and suggest that the novel competing endogenous motifs play important roles in expression regulation and could be potential target for investigating ovarian cancer mechanism or its therapy.Peer reviewe

    CLIP and complementary methods

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    RNA molecules start assembling into ribonucleoprotein (RNP) complexes during transcription. Dynamic RNP assembly, largely directed by cis-acting elements on the RNA, coordinates all processes in which the RNA is involved. To identify the sites bound by a specific RNA-binding protein on endogenous RNAs, cross-linking and immunoprecipitation (CLIP) and complementary, proximity-based methods have been developed. In this Primer, we discuss the main variants of these protein-centric methods and the strategies for their optimization and quality assessment, as well as RNA-centric methods that identify the protein partners of a specific RNA. We summarize the main challenges of computational CLIP data analysis, how to handle various sources of background and how to identify functionally relevant binding regions. We outline the various applications of CLIP and available databases for data sharing. We discuss the prospect of integrating data obtained by CLIP with complementary methods to gain a comprehensive view of RNP assembly and remodelling, unravel the spatial and temporal dynamics of RNPs in specific cell types and subcellular compartments and understand how defects in RNPs can lead to disease. Finally, we present open questions in the field and give directions for further development and applications

    Investigating the Prevalence of RNA-Binding Metabolic Enzymes in E. coli

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    An open research field in cellular regulation is the assumed crosstalk between RNAs, metabolic enzymes, and metabolites, also known as the REM hypothesis. High-throughput assays have produced extensive interactome data with metabolic enzymes frequently found as hits, but only a few examples have been biochemically validated, with deficits especially in prokaryotes. Therefore, we rationally selected nineteen Escherichia coli enzymes from such datasets and examined their ability to bind RNAs using two complementary methods, iCLIP and SELEX. Found interactions were validated by EMSA and other methods. For most of the candidates, we observed no RNA binding (12/19) or a rather unspecific binding (5/19). Two of the candidates, namely glutamate-5-kinase (ProB) and quinone oxidoreductase (QorA), displayed specific and previously unknown binding to distinct RNAs. We concentrated on the interaction of QorA to the mRNA of yffO, a grounded prophage gene, which could be validated by EMSA and MST. Because the physiological function of both partners is not known, the biological relevance of this interaction remains elusive. Furthermore, we found novel RNA targets for the MS2 phage coat protein that served us as control. Our results indicate that RNA binding of metabolic enzymes in procaryotes is less frequent than suggested by the results of high-throughput studies, but does occur

    Characterisation of RNA binding proteins and their roles in the Drosophila germline

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    The important role of RNA binding proteins (RBPs) in regulating the fate and functions of RNAs has led to the development of transcript-specific as well as transcriptome-wide techniques allowing an unbiased and comprehensive identification of RBPs. These methods have extended our knowledge of the extent of RBPs in a cell, and studying the roles of these newly identified RBPs in cellular processes has provided us with novel insights into the RNA binding mechanisms, functions and regulation of RNA binding proteins. For my PhD work, I assessed the RNA binding functions of two proteins identified in high-throughput screens. The first protein is the Fragile X Mental Retardation protein (FMR1), identified in a transcript-specific pulldown targeted at the Drosophila maternal mRNA oskar. I show that FMR1 is a bona fide component of the oskar RNA-protein complexes that interacts with the oskar 3’UTR in vivo. FMR1 positively regulates Oskar protein levels in the oocyte, without any effect on oskar RNA levels. Oskar protein nucleates germ plasm assembly and germ cell formation in the embryo, and the reduction in Oskar protein levels leads to a reduction in the number of pole cells formed in embryos knocked down for FMR1. Finally, I tried to determine how FMR1 regulates translation, with roles identified as both a repressor and activator of translation. FMR1 contains two types of RNA binding domains: two KH domains and a C-terminal RGG box. I show that, in vitro, FMR1 activates translation through the KH domains and requires the C-terminal RGG box for repression of translation. I have thus identified a new role of FMR1 in germline development in Drosophila melanogaster, and also a putative mechanism of how FMR1 performs antagonistic functions in translation regulation. The second protein I studied is the microtubule binding protein EB1, identified as a putative RNA binding protein in a transcriptome-wide RNA interactome capture study performed in Drosophila embryos. Preliminary data showed that EB1 binds to polyU25 RNA in vitro, and uses the same binding surface for interacting with microtubules and RNA. I show that EB1 binds to microtubules and RNA in a mutually exclusive manner in vitro. Furthermore, I performed a RIP-seq experiment to identify the in vivo targets of EB1, but failed to validate the interaction of any of the top candidates with EB1 in vivo. This does not, however, negate a role of EB1 as an RNA binding protein altogether, as RNA might be regulating the functions of the protein, and this would require further investigation

    System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organization and function of RNA–protein complexes

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    Large RNA-binding complexes play a central role in gene expression and orchestrate production, function, and turnover of mRNAs. The accuracy and dynamics of RNA–protein interactions within these molecular machines are essential for their function and are mediated by RNA-binding proteins (RBPs). Here, we show that fission yeast whole-cell poly(A)+ RNA–protein crosslinking data provide information on the organization of RNA–protein complexes. To evaluate the relative enrichment of cellular RBPs on poly(A)+ RNA, we combine poly(A)+ RNA interactome capture with a whole-cell extract normalization procedure. This approach yields estimates of in vivo RNA-binding activities that identify subunits within multiprotein complexes that directly contact RNA. As validation, we trace RNA interactions of different functional modules of the 3′ end processing machinery and reveal additional contacts. Extending our analysis to different mutants of the RNA exosome complex, we explore how substrate channeling through the complex is affected by mutation. Our data highlight the central role of the RNA helicase Mtl1 in regulation of the complex and provide insights into how different components contribute to engagement of the complex with substrate RNA. In addition, we characterize RNA-binding activities of novel RBPs that have been recurrently detected in the RNA interactomes of multiple species. We find that many of these, including cyclophilins and thioredoxins, are substoichiometric RNA interactors in vivo. Because RBPomes show very good overall agreement between species, we propose that the RNA-binding characteristics we observe in fission yeast are likely to apply to related proteins in higher eukaryotes as well

    Functional characterization of PAC and PUMPKIN, two proteins involved in chloroplast RNA metabolism

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    lincRNAs: Genomics, Evolution, and Mechanisms

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    Long intervening noncoding RNAs (lincRNAs) are transcribed from thousands of loci in mammalian genomes and might play widespread roles in gene regulation and other cellular processes. This Review outlines the emerging understanding of lincRNAs in vertebrate animals, with emphases on how they are being identified and current conclusions and questions regarding their genomics, evolution and mechanisms of action.National Institutes of Health (U.S.) (Grant GM067031

    The Mystery of Nuclear Localization of AROGENATE DEHYDRATASE5 from Arabidopsis thaliana

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    Arogenate dehydratases (ADTs) have been identified to catalyze the last step of phenylalanine (Phe) biosynthesis in plants. All ADTs have a transit peptide sequence that targets them into the chloroplasts where the biosynthesis of Phe happens. Subcellular localization studies using fluorescently tagged Arabidopsis thaliana ADTs demonstrated that all six ADTs localize to chloroplast stromules (stroma filled tubules). However, one member of this family, ADT5, was also detected in the nucleus. As dual targeting of proteins to different cell compartments is an indication of multifunctionality, ADT5 nuclear localization suggests that this member of the ADT protein family is a moonlighting protein with a non-enzymatic role in the nucleus. In this study, first the nuclear localization of the ADT5 was confirmed by expression of ADT5-CFP under the regulation of ADT5 native promoter. Using confocal microscopy and Western blot analysis it was shown that ADT5 localizes into the nucleus. Next, different possible mechanisms that could result to the nuclear localization of ADT5 were studied. It was tested if ADT5 can move directly from chloroplast stroma to the nucleus through stromules or if ADT5 enter the nucleus from cytoplasm using the nuclear import system. Data presented are consistent with a translocation from cytoplasm. A combination of an AQEH motif and a single amino acid Asn28 present in the N-terminus of ADT5 ACT domain were identified as potential protein interaction sites required for nuclear targeting of ADT5. Y2H screenings and protein-protein interaction analyses suggest that nuclear localization of ADT5 occurs through the interaction of the cytosolic portion of an ER membrane bound protein, PHOSPHOLIPID DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1). A nuclear targeting sequence was identified in the N-terminal cytosolic portion of PDAT1. Hence, it is possible that PDAT1 piggybacks ADT5 into the nucleus through the nuclear import system. This study introduces ADT5 as a moonlighting protein and identifies a possible mechanism for ADT5 nuclear translocation
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