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A versatile IRES toolbox to determine IRES-like activity exemplified by Hoxa mRNA expression and translation in mouse embryonic tissues

ABSTRACT: A versatile IRES toolbox to determine IRES-like activity exemplified by Hoxa mRNA expression and translation in mouse embryonic tissues

PROVIDER: PRJNA1052683 | ENA |

REPOSITORIES: ENA

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	SRR27215150_subreads.fastq.gz	Fastqsanger.gz

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A versatile IRES toolbox to determine IRES-like activity exemplified by Hoxa mRNA expression and translation in mouse embryonic tissues

Project description:Widespread control of gene expression at the level of translation has emerged as a key point of protein expression regulation in space and time. Internal ribosomal entry sites (IRESes) are a prominent mechanism by which ribosomes can confer greater gene regulation. However, their rigorous functional characterization remains difficult. Here we present a versatile toolbox of technologies in embryos and cells including single-molecule mRNA isoform imaging, Pacbio long read sequencing, isoform-sensitive mRNA quantification along polysome profiles, and IRES-like translation of circular RNA (circRNA) reporters as a new guide to understanding IRES-mediated regulation. We investigate the disputed IRES-like RNA elements in embryonic Hoxa mRNAs. We show the relative expression, localization and translation of the IRES-like containing Hoxa9 mRNA isoform in specific embryonic tissues, and Hoxa IRES-like dependent translation in circRNAs. We thereby provide a new resource of technologies to elucidate the roles of IRES-like elements in gene regulation and embryonic development.

2025-03-06 | GSE250214 | GEO

Pectobacterium versatile

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Identification of the TXNIP IRES and characterization of the impact of regulatory IRES trans-acting factors

Project description:Translation is a tightly regulated and is predominantly controlled at the level of its initiation. Initiation occurs in a cap-dependent manner. Under stress conditions when cap-dependent translation is hampered, internal ribosome entry sites (IRESes) allow for cap-independent translation of certain mRNAs. IRES-dependent translation is commonly regulated by RNA-interacting proteins, known as IRES trans-acting factors (ITAFs). In the present study, we searched for new IRESes by identifying 5’ untranslated regions (UTRs) bound by the ITAF hnRNPA1. Using a PAR-iCLIP approach, we found the mRNA of thioredoxin interacting protein (TXNIP) bound by hnRNPA1. Upon verification of an IRES element within the 5’UTR of TXNIP, we determined additional interacting proteins, which predominantly appeared to interact with the IRES-regulatory second half of the 5’UTR. Amongst these PTB, FBP3, and GEMIN5 emerged as functional ITAFs. Finally, we found that the TXNIP IRES-inhibitory effect of PTB was dominant over the activating effect of FBP3, while it succumbed to the stimulatory function of GEMIN5. In summary, we identified and characterized a novel IRES within the 5’UTR of TXNIP, which is regulated by the ITAFs PTB, FBP3, and GEMIN5.

2018-01-23 | GSE97159 | GEO

Pectobacterium versatile

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Deletion of the Hoxa Cluster in MLL-AF9 Is Incompatible with Leukemia Maintenance: RNAseq-based Quantitative Analysis of Wild Type and Hoxa-del Transcriptomes

Project description:High HOXA expression correlates with poor clinical outcome in AML, particularly those harboring MLL rearrangements (MLLr). The necessity of the HOXA cluster for the maintenance of MLLr-leukemia has not been elucidated. Primary leukemias were generated by transduction of MLL-AF9 (MA9) into hematopoietic stem and progenitor cells from compound Cre responsive transgenic mice for conditional deletion of the Hoxa locus. Hoxa deletion resulted in reduced proliferation, colony formation and repopulating ability in transplanted mice in which surviving leukemic cells retained at least one copy of the Hoxa cluster (Hoxa-del). Comparative RNA-seq analysis of leukemic MA9-Hoxa-wild type (WT) and MA9-Hoxa-del cells identified a unique gene signature.

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Identification of human FGF9 IRES interacting proteins

Project description:We have previously shown that FGF9 is overexpressed in hypoxia through the IRES located in the 5’UTR. To identify the protein that binds to FGF9 IRES and controls FGF9 protein synthesis in hypoxia, FGF9 IRES RNA was in vitro synthesized and used to pulled-down interacting proteins. The RNA-protein complexes were first visualized by sliver staining, followed by cutting specific bands for protein identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS).

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IRES containing mRNAs and mitotic cell cycle

Project description:A cell synchronization protocol was established in which global and individual mRNA translational efficiencies could be examined. While global translational efficiency was reduced in mitotic cells, approximately 3% of mRNAs remained predominantly associated with large polysomes during mitosis, as determined by cDNA microarray analyses. The 5 noncoding regions of six mRNAs were shown to contain internal ribosome entry sites (IRES). However, not all known mRNAs that contain IRES elements were actively translated during mitosis, arguing that specific IRES sequences are differentially regulated during mitosis. A cell cycle design experiment design type is one that assays events that occurs in relation to the cell cycle, which is the period between the formation of a cell, by division of its mother cell and the time when the cell itself divides to form two daughter cells. Keywords: cell_cycle_design

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A toolbox for epitope-tagging and genome-wide location analysis in Candida albicans

Project description:Background : Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent. Results: We have developed a toolbox allowing in vivo protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of C. albicans Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with in vivo TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for in vivo analysis of transcriptional activity of gene loci in C. albicans. Conclusion: This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in C. albicans. Two independent biological replicates of ChIP-CHIP of Mcm1-TAP in yeast and hyphal states. ChIP-CHIP of Cbf1-TAP and Tbf1-TAP.

2010-05-16 | E-GEOD-13427 | biostudies-arrayexpress

Identification of ribosome proteins bound to IRES in hypoxia

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