Project description:MicroRNAs (miRNAs) are small RNAs that function as post-transcriptional regulators of gene expression. miRNAs affect a variety of signaling pathways and impaired miRNA regulation may contribute to the development of cancer and other diseases. We show that miRNA miR-10a interacts with the 5' untranslated region of mRNAs encoding ribosomal proteins and enhances their translation. miR-10a alleviates translational repression of the ribosomal protein mRNAs during amino acid starvation and is required for their stress-mediated activation following anisomycin treatment. miR-10a binds immediately downstream of the regulatory 5' TOP motif and the 5´TOP is necessary for miR-10a translational enhancement. The results indicate that miR-10a may positively control global protein synthesis via stimulation of ribosomal protein mRNA translation and that the 5' TOP regulatory complex and miR-10a are functionally interconnected.
Project description:Precise spatiotemporal control of mRNA translation machinery is essential to proper development of highly complex systems like the neocortex. Here, we show that an RNA-binding protein, Hu antigen R (HuR), regulates both neocorticogenesis and specificity of neocortical translation machinery in a developmental stagedependent manner in mice. Neocortical absence of HuR alters the phosphorylation states of the initiation and elongation factors of the core translation machinery. In addition, HuR regulates the temporally specific positioning of functionally related mRNAs into the active translation sites, the polysomes. HuR also determines the specificity of neocortical polysomes by defining their combinatorial composition of ribosomal proteins and initiation and elongation factors. For some of the HuR-dependent proteins, the association with polysomes depends on the eIF2 alpha kinase 4 (eIF2ak4), which associated with HuR in prenatal developing neocortices. Finally, we found that deletion of HuR prior to embryonic day 10 (E10) disrupts both neocortical lamination and formation of the main neocortical commissure, the corpus callosum. Our study identifies a crucial role for HuR in neocortical development as a translational gatekeeper for functionally related mRNA subgroups and polysomal protein specificity. Cortex was dissected from mouse pups at embryonic day 13 (E13) or the day of birth (P0).
Project description:Duplicated genes escape gene loss by conferring a dosage benefit or evolving diverged functions. The yeast Saccharomyces cerevisiae contains many duplicated genes encoding ribosomal proteins. Prior studies have suggested that these duplicated proteins are functionally redundant and affect cellular processes in proportion to their expression. In contrast, through studies of ASH1 mRNA in yeast, we demonstrate paralog-specific requirements for the translation of localized mRNAs. Intriguingly, these paralog-specific effects are limited to a distinct subset of duplicated ribosomal proteins. Moreover, transcriptional and phenotypic profiling of cells lacking specific ribosomal proteins reveals differences between the functional roles of ribosomal protein paralogs that extend beyond effects on mRNA localization. Finally, we show that ribosomal protein paralogs exhibit differential requirements for assembly and localization. Together, our data indicate complex specialization of ribosomal proteins for specific cellular processes, and support the existence of a ribosomal code. Experiment Overall Design: We used Affymetrix arrays to analyze the transcriptional profiles of cells lacking certain duplicated ribosomal protein genes in order to determine if paralogous ribosomal proteins have differing cellular effects and roles. Experiment Overall Design: Two biological replicates were performed for each strain. Each ribosomal protein deletion was then compared to the isogenic wild-type strain.
Project description:Targeted depletion of ribosomal protein S6 (Rps6) from hepatoblasts of the developing liver results in neonatal sub-lethal hepatic failure due to inhibition of bile duct development and widespread induction of hepatocyte death triggering regeneration. Overexpression of c-Myc is hepatoprotective in the context of Rps6-deficiency and eliminates the need for DS6 livers to regenerate by preventing hepatocyte death. Microarrays were used to identify the transcriptional program associated with loss of hepatic Rps6 and to identify mRNAs associated with c-Myc's hepatoprotective effect in DS6 livers
Project description:RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurologic disease. Developing axons provide a well suited model to study RNA-based regulation, and contain specific subsets of mRNAs that are locally translated and have roles in axon pathfinding. However, the RNA-binding proteins involved in axon pathfinding, and their corresponding mRNA targets, are still largely unknown. Here we find that the RNA-binding protein IMP2 (Igf2bp2) is strikingly enriched in developing axon tracts, including in spinal commissural axons. We used the HITS-CLIP approach to perform a genome-wide identification of RNAs that interact directly with IMP2 in the native context of developing brain. This IMP2 interactome was highly enriched for mRNA targets related to axon guidance. Accordingly, IMP2 knockdown in the developing spinal cord led to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal its requirement for normal axon pathfinding during vertebrate development. CLIP-seq
Project description:Duplicated genes escape gene loss by conferring a dosage benefit or evolving diverged functions. The yeast Saccharomyces cerevisiae contains many duplicated genes encoding ribosomal proteins. Prior studies have suggested that these duplicated proteins are functionally redundant and affect cellular processes in proportion to their expression. In contrast, through studies of ASH1 mRNA in yeast, we demonstrate paralog-specific requirements for the translation of localized mRNAs. Intriguingly, these paralog-specific effects are limited to a distinct subset of duplicated ribosomal proteins. Moreover, transcriptional and phenotypic profiling of cells lacking specific ribosomal proteins reveals differences between the functional roles of ribosomal protein paralogs that extend beyond effects on mRNA localization. Finally, we show that ribosomal protein paralogs exhibit differential requirements for assembly and localization. Together, our data indicate complex specialization of ribosomal proteins for specific cellular processes, and support the existence of a ribosomal code. Keywords: genetic modification
Project description:Duplicated genes escape gene loss by conferring a dosage benefit or evolving diverged functions. The yeast Saccharomyces cerevisiae contains many duplicated genes encoding ribosomal proteins. Prior studies have suggested that these duplicated proteins are functionally redundant and affect cellular processes in proportion to their expression. In contrast, through studies of ASH1 mRNA in yeast, we demonstrate paralog-specific requirements for the translation of localized mRNAs. Intriguingly, these paralog-specific effects are limited to a distinct subset of duplicated ribosomal proteins. Moreover, transcriptional and phenotypic profiling of cells lacking specific ribosomal proteins reveals differences between the functional roles of ribosomal protein paralogs that extend beyond effects on mRNA localization. Finally, we show that ribosomal protein paralogs exhibit differential requirements for assembly and localization. Together, our data indicate complex specialization of ribosomal proteins for specific cellular processes, and support the existence of a ribosomal code. Keywords: genetic modification
Project description:The initiation of translation begins with formation of a ribosome-mRNA complex. In bacteria, the 30S ribosomal subunit is recruited to many mRNAs through both base pairing with the Shine Dalgarno (SD) sequence and RNA-binding by ribosomal protein bS1. Translation can initiate on mRNAs that are being transcribed, and RNA polymerase (RNAP) can promote recruitment of the pioneering 30S. Here we have examined ribosome recruitment to mRNAs using cryogenic electron microscopy (cryo-EM), single-molecule fluorescence co-localization, and whole-cell cross-linking mass spectrometry. Structures of 30S-mRNA complexes show that bS1 delivers the mRNA to the ribosome for SD duplex formation and initial 30S subunit activation. RNAP associates flexibly with the 30S platform during nascent mRNA delivery and accelerates their association in a bS1-dependent manner in vitro. Collectively, our data provide a mechanistic framework for how the SD duplex, ribosomal proteins and RNAP cooperate in 30S recruitment to mRNAs and thereby establish transcription-translation coupling.