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Basic research in Drosophila melanogaster has benefited from a plethora of powerful genetics tools. Detailed biochemical analysis, however, has often been difficult due to the lack of in vitro systems that faithfully recapitulate the observations made in vivo. In the field of posttranscriptional regulation, the recent establishment of robust in vitro systems from embryo and ovary material has fueled the mechanistic understanding of a variety of processes. Here we describe protocols to obtain and use extracts from Drosophila embryos that are competent for cytoplasmic polyadenylation and translation of exogenously added transcripts.
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http://dx.doi.org/10.1007/978-1-62703-971-0_5 | DOI Listing |
Nucleic Acids Res
February 2025
Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, United States.
Poly(A)-binding protein (Pab1 in yeast) is involved in mRNA decay and translation initiation, but its molecular functions are incompletely understood. We found that auxin-induced degradation of Pab1 reduced bulk mRNA and polysome abundance in WT but not in a mutant lacking the catalytic subunit of decapping enzyme (Dcp2), suggesting that enhanced decapping/degradation is a major driver of reduced translation at limiting Pab1. An increased median poly(A) tail length conferred by Pab1 depletion was likewise not observed in the dcp2Δ mutant, suggesting that mRNA isoforms with shorter tails are preferentially decapped/degraded at limiting Pab1.
View Article and Find Full Text PDFNucleic Acids Res
February 2025
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.
Eukaryotic translation initiation factor 4F (eIF4F), comprising subunits eIF4G, eIF4E, and eIF4A, plays a pivotal role in the 48S preinitiation complex assembly and ribosomal scanning. Additionally, eIF4B enhances the helicase activity of eIF4A. eIF4F also interacts with poly (A)-binding protein (PABP) bound to the poly (A) tail of messenger RNA (mRNA), thereby forming a closed-loop structure.
View Article and Find Full Text PDFBiochimie
February 2025
Sorbonne Université, Institute of Biology Paris-Seine, IBPS, CNRS, UMR 8256, Biological Adaptation and Ageing, B2A, F 75005, Paris, France; Sorbonne Université, Institute of Biology Paris-Seine, IBPS, CNRS, UMR 8263, INSERM U1345 Development Adaptation and Ageing, Dev2A, F 75005, Paris, France. Electronic address:
In humans, the release factor eRF3a exists in several forms that differ in the length of the polyglycine tract (7, 10, 11 or 12 glycines) in its N-terminal domain. For the 12-Gly eRF3a, an association with cancer risk and a decreased affinity for the cytoplasmic poly (A) binding protein have already been established. In this work, HCT116 colon cancer cells were treated with low doses of doxorubicin, which is known to induce senescence in these cells with high efficiency.
View Article and Find Full Text PDFmBio
February 2025
Department of Microbiology and Molecular Genetics, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
Unlabelled: Human immunodeficiency virus type 1 (HIV-1) capsid, which is the target of the antiviral lenacapavir, protects the viral genome and binds multiple host proteins to influence intracellular trafficking, nuclear import, and integration. Previously, we showed that capsid binding to cleavage and polyadenylation specificity factor 6 (CPSF6) in the cytoplasm is competitively inhibited by cyclophilin A (CypA) binding and regulates capsid trafficking, nuclear import, and infection. Here, we determined that a capsid mutant with increased CypA binding affinity had significantly reduced nuclear entry and mislocalized integration.
View Article and Find Full Text PDFMol Med
February 2025
Department of Neurosurgery, The Second Affiliated Hospital of Harbin Medical University, No. 246, Xuefu Road, Nangang District, Harbin, Heilongjiang, 150001, China.
Background: The pathogenesis of epilepsy is complex, and current antiepileptic drugs do not effectively control the seizures. Cytoplasmic polyadenylation element-binding protein 3 (CPEB3) regulates neuronal excitability, but its mechanism of action in epilepsy is not clear. In this paper, we investigated the effect of CPEB3 on seizures and elucidated its underlying molecular mechanism.
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