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1.
Nat Commun ; 15(1): 5270, 2024 Jun 20.
Article in English | MEDLINE | ID: mdl-38902233

ABSTRACT

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent mRNA stability in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for mRNA stability and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA stability and protein expression.


Subject(s)
Cell Differentiation , Drosophila Proteins , Neural Stem Cells , Neurons , RNA Stability , RNA, Messenger , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Neurons/metabolism , Neurons/cytology , RNA, Messenger/metabolism , RNA, Messenger/genetics , Cell Differentiation/genetics , Neural Stem Cells/metabolism , Neural Stem Cells/cytology , Codon/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Receptors, Metabotropic Glutamate/metabolism , Receptors, Metabotropic Glutamate/genetics , mRNA Cleavage and Polyadenylation Factors/metabolism , mRNA Cleavage and Polyadenylation Factors/genetics , Drosophila/genetics , Drosophila/metabolism , Brain/metabolism , Brain/cytology , Transcription Factors
2.
bioRxiv ; 2023 Jul 27.
Article in English | MEDLINE | ID: mdl-37546801

ABSTRACT

Regulation of codon optimality is an increasingly appreciated layer of cell- and tissue-specific protein expression control. Here, we use codon-modified reporters to show that differentiation of Drosophila neural stem cells into neurons enables protein expression from rare-codon-enriched genes. From a candidate screen, we identify the cytoplasmic polyadenylation element binding (CPEB) protein Orb2 as a positive regulator of rare-codon-dependent expression in neurons. Using RNA sequencing, we reveal that Orb2-upregulated mRNAs in the brain with abundant Orb2 binding sites have a rare-codon bias. From these Orb2-regulated mRNAs, we demonstrate that rare-codon enrichment is important for expression control and social behavior function of the metabotropic glutamate receptor (mGluR). Our findings reveal a molecular mechanism by which neural stem cell differentiation shifts genetic code regulation to enable critical mRNA and protein expression.

3.
Elife ; 112022 05 06.
Article in English | MEDLINE | ID: mdl-35522036

ABSTRACT

Codon usage bias has long been appreciated to influence protein production. Yet, relatively few studies have analyzed the impacts of codon usage on tissue-specific mRNA and protein expression. Here, we use codon-modified reporters to perform an organism-wide screen in Drosophila melanogaster for distinct tissue responses to codon usage bias. These reporters reveal a cliff-like decline of protein expression near the limit of rare codon usage in endogenously expressed Drosophila genes. Near the edge of this limit, however, we find the testis and brain are uniquely capable of expressing rare codon-enriched reporters. We define a new metric of tissue-specific codon usage, the tissue-apparent Codon Adaptation Index (taCAI), to reveal a conserved enrichment for rare codon usage in the endogenously expressed genes of both Drosophila and human testis. We further demonstrate a role for rare codons in an evolutionarily young testis-specific gene, RpL10Aa. Optimizing RpL10Aa codons disrupts female fertility. Our work highlights distinct responses to rarely used codons in select tissues, revealing a critical role for codon bias in tissue biology.


Subject(s)
Drosophila melanogaster , Drosophila , Animals , Codon/genetics , Codon Usage , Drosophila/genetics , Drosophila melanogaster/genetics , Female , Humans , Male , Testis
4.
PLoS Genet ; 16(12): e1009228, 2020 12.
Article in English | MEDLINE | ID: mdl-33296356

ABSTRACT

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.


Subject(s)
Codon Usage , Drosophila Proteins/genetics , Genes, Modifier , Mitogen-Activated Protein Kinases/genetics , ras Proteins/genetics , Animals , Drosophila Proteins/metabolism , Drosophila melanogaster , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , ras Proteins/metabolism
5.
J Neurosci ; 39(32): 6233-6250, 2019 08 07.
Article in English | MEDLINE | ID: mdl-31182634

ABSTRACT

Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation.SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules.


Subject(s)
Cell Adhesion Molecules/physiology , Dendritic Spines/physiology , Prefrontal Cortex/physiology , Semaphorins/physiology , Visual Cortex/physiology , Aging/physiology , Animals , Cell Adhesion Molecules/deficiency , Cells, Cultured , Female , GABA Agonists/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nerve Tissue Proteins/physiology , Neuropilin-2/physiology , Patch-Clamp Techniques , Prefrontal Cortex/cytology , Prefrontal Cortex/growth & development , Protein Interaction Mapping , Pyramidal Cells/drug effects , Pyramidal Cells/physiology , Pyramidal Cells/ultrastructure , Pyridazines/pharmacology , Receptors, Cell Surface/physiology , Synaptic Transmission , Visual Cortex/cytology , Visual Cortex/growth & development
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