scholarly journals miR-375 Inhibits Differentiation of Neurites by Lowering HuD Levels

2010 ◽  
Vol 30 (17) ◽  
pp. 4197-4210 ◽  
Author(s):  
Kotb Abdelmohsen ◽  
Emmette R. Hutchison ◽  
Eun Kyung Lee ◽  
Yuki Kuwano ◽  
Mihee M. Kim ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Neuronal Development ◽  
Rna Binding ◽  
Developmentally Regulated ◽  
Cytoskeleton Organization ◽  
Evolutionarily Conserved ◽  
Mouse Hippocampus ◽  
Regulated Gene Expression ◽  
And Function

ABSTRACT Neuronal development and plasticity are maintained by tightly regulated gene expression programs. Here, we report that the developmentally regulated microRNA miR-375 affects dendrite formation and maintenance. miR-375 overexpression in mouse hippocampus potently reduced dendrite density. We identified the predominantly neuronal RNA-binding protein HuD as a key effector of miR-375 influence on dendrite maintenance. Heterologous reporter analysis verified that miR-375 repressed HuD expression through a specific, evolutionarily conserved site on the HuD 3′ untranslated region. miR-375 overexpression lowered both HuD mRNA stability and translation and recapitulated the effects of HuD silencing, which reduced the levels of target proteins with key functions in neuronal signaling and cytoskeleton organization (N-cadherin, PSD-95, RhoA, NCAM1, and integrin α1). Moreover, the increase in neurite outgrowth after brain-derived neurotrophic factor (BDNF) treatment was diminished by miR-375 overexpression; this effect was rescued by reexpression of miR-375-refractory HuD. Our findings indicate that miR-375 modulates neuronal HuD expression and function, in turn affecting dendrite abundance.

scholarly journals Immunohistochemical Characterization of Phosphorylated Ubiquitin in the Mouse Hippocampus

2020 ◽  
Author(s):  
Kosuke Kataoka ◽  
Andras Bilkei-Gorzo ◽  
Andreas Zimmer ◽  
Toru Asahi
Keyword(s):  
Cell Layer ◽  
Granule Cell Layer ◽  
Phosphatase And Tensin Homolog ◽  
Neuronal Markers ◽  
Tensin Homolog ◽  
Evolutionarily Conserved ◽  
Previous Hypothesis ◽  
Mouse Hippocampus ◽  
And Function

ABSTRACTMitochondrial autophagy (mitophagy) is an essential and evolutionarily conserved process that maintains mitochondrial integrity via the removal of damaged or superfluous mitochondria in eukaryotic cells. Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and Parkin promote mitophagy and function in a common signaling pathway. PINK1-mediated ubiquitin phosphorylation at Serine 65 (Ser65-pUb) is a key event in the efficient execution of PINK1/Parkin-dependent mitophagy. However, few studies have used immunohistochemistry to analyze Ser65-pUb in the mouse. Here, we examined the immunohistochemical characteristics of Ser65-pUb in the mouse hippocampus. Some hippocampal cells were Ser65-pUb positive, whereas the remaining cells expressed no or low levels of Ser65-pUb. PINK1 deficiency resulted in a decrease in the density of Ser65-pUb-positive cells, consistent with a previous hypothesis based on in vitro research. Interestingly, Ser65-pUb-positive cells were detected in hippocampi lacking PINK1 expression. The CA3 pyramidal cell layer and the dentate gyrus (DG) granule cell layer exhibited significant reductions in the density of Ser65-pUb-positive cells in PINK1-deficient mice. Moreover, Ser65-pUb immunoreactivity colocalized predominantly with neuronal markers. These findings suggest that Ser65-pUb may serve as a biomarker of in situ PINK1 signaling in the mouse hippocampus; however, the results should be interpreted with caution, as PINK1 deficiency downregulated Ser65-pUb only partially.

scholarly journals The Class I E3 Ubiquitin Ligase TRIM67 Modulates Brain Development and Behavior

2017 ◽  
Author(s):  
Nicholas P. Boyer ◽  
Caroline Monkiewicz ◽  
Sheryl S. Moy ◽  
Stephanie L. Gupton
Keyword(s):  
Brain Development ◽  
Axon Guidance ◽  
Neuronal Development ◽  
Sensorimotor Gating ◽  
Brain Regions ◽  
Class I ◽  
Guidance Cue ◽  
Evolutionarily Conserved ◽  
And Function ◽  
And Behavior

ABSTRACTSpecific class I members of the TRIM family of E3 ubiquitin ligases have been implicated in neuronal development from invertebrates to mammals. The single invertebrate class I TRIM and mammalian TRIM9 regulate axon branching and guidance in response to the axon guidance cue netrin-1, whereas mammalian TRIM46 establishes the axon initial segment. In humans, mutations in TRIM1 and TRIM18 are implicated in Optiz Syndrome, characterized by midline defects and often mild intellectual disability. TRIM67 is the most evolutionarily conserved vertebrate class I TRIM, yet is the least studied. Here we show that TRIM67 interacts with both its closest paralog TRIM9 and the netrin receptor DCC, and is differentially enriched in specific brain regions at specific developmental points. We describe the anatomical and behavioral consequences of deletion of murine Trim67. While viable, mice lacking Trim67 display severe impairments in spatial memory, cognitive flexibility, social novelty preference, muscle function and sensorimotor gating. Additionally, they exhibit abnormal anatomy of several brain regions, including the hippocampus, striatum and thalamus, as well as the corpus callosum. This study demonstrates the necessity for TRIM67 in appropriate brain development and function.SIGNIFICANCE STATEMENTAs a family, class I TRIM E3 ubiquitin ligases play important roles in neuronal development and function, potentially cooperatively. TRIM67 is the most evolutionarily conserved class I TRIM and is developmentally regulated and brain-enriched. Deletion of murine Trim67 results in malformations of a subset subcortical brain regions and of cortical and subcortical myelinated fiber tracts, as well as deficits in spatial memory, motor function, sociability and sensorimotor gating. We conclude that TRIM67 is critical for appropriate brain development and behavior, potentially downstream of the axon guidance cue netrin, and in cooperation with class I TRIM9.

scholarly journals A Nucleolin-Binding 3′ Untranslated Region Element Stabilizes β-Globin mRNA In Vivo

2006 ◽  
Vol 26 (6) ◽  
pp. 2419-2429 ◽  
Author(s):  
Yong Jiang ◽  
Xiang-Sheng Xu ◽  
J. Eric Russell
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Rna Binding ◽  
Critical Role ◽  
Specific Interaction ◽  
Globin Mrna ◽  
Intact Cells ◽  
Normal Expression

ABSTRACT The normal expression of human β globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with α-globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing β-globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the β-globin 3′ untranslated region (3′UTR). The new determinant is positioned on an mRNA half-stem opposite a pyrimidine-rich sequence targeted by αCP/hnRNP-E, a factor that plays a critical role in stabilizing human α-globin mRNA. Mutations within the new determinant destabilize β-globin mRNA in intact cells while also ablating its 3′UTR-specific interaction with the polyfunctional RNA-binding factor nucleolin. We speculate that 3′UTR-bound nucleolin enhances mRNA stability by optimizing αCP access to its functional binding site. This model is favored by in vitro evidence that αCP binding is enhanced both by cis-acting stem-destabilizing mutations and by the trans-acting effects of supplemental nucleolin. These studies suggest a mechanism for β-globin mRNA stability that is related to, but distinct from, the mechanism that stabilizes human α-globin mRNA.

scholarly journals Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

1998 ◽  
Vol 9 (2) ◽  
pp. 469-481 ◽  
Author(s):  
Kevin P. Claffey ◽  
Shu-Ching Shih ◽  
Andrew Mullen ◽  
Suzan Dziennis ◽  
Jennifer L. Cusick ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Stability Region ◽  
Rna Binding ◽  
Malignant Tumors ◽  
Human Tumor ◽  
Luciferase Reporter ◽  
Vegf Expression ◽  
Vegf Mrna ◽  
Vascular Permeability Factor

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.

scholarly journals Application of the 3'-Untranslated Region of Messenger RNA from Measles Virus Matrix Protein as an RNA Stabilizer: Implications in Pharmaceutical Biotechnology

2019 ◽  
Vol 9 (1) ◽  
pp. 53
Author(s):  
Marzieh Marzbany ◽  
Fatemeh Ghassemi ◽  
Mahsa Rasekhian
Keyword(s):  
Mrna Stability ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Measles Virus ◽  
Matrix Protein ◽  
Rna Binding ◽  
Rna Stability ◽  
Minimum Free Energy ◽  
Expression Vectors ◽  
Dependent Manner

BACKGROUND: The market for the use of recombinant proteins for medical applications has been increasing in recent years. In many cases including fast production of significant amounts of protein for research purposes, transient transfection is the method of choice. In this regard expression vectors are one of the decisive factors in the cost-effectiveness of the production process. The genetic elements found in the 3’untranslated region (UTR) of mRNA expressed by such vectors, play an essential role in determining its stability and thus in the efficiency of the process. METHODS: In this study, the 3'UTR of matrix protein from the Measles Virus (MV) was used to construct a reporter plasmid containing Enhanced Green Fleurocent Protein (EGFP). The reporter construct was transfected into three cell lines. The effect of 3'UTR on mRNA stability was evaluated by real-time PCR. Secondary structure of the mrna was predicted based on minimum free energy. 3'UTR was analyzed in silico for the presence of binding motifs for trans-acting elements with known effects on RNA stability. RESULTS: Addition of 3’UTR of MV matrix protein sequence to the 3’ end of the mRNA, increased the EGFP- mRNA stability in time and cell-dependent manner. Analysis for the presence of known cis-acting motifs in 3’UTR indicated the presence of two PABPC1 binding sites, an RNA-binding protein, known for its stability and translation enhancing effects. CONCLUSION: Our results verified the potential of the 3'UTR region of matrix protein mRNA for improvement of transient recombinant protein production and vector design for mammalian cell hosts.

FMRP and MicroRNAs in Neuronal Protein Synthesis

2018 ◽  
Author(s):  
Monica C. Lannom ◽  
Stephanie Ceman
Keyword(s):  
Protein Synthesis ◽  
Binding Proteins ◽  
Neuronal Development ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Post Translational Modifications ◽  
Local Protein Synthesis ◽  
Recognition Element ◽  
Neuronal Protein ◽  
And Function

New protein synthesis is critical for learning and memory. The discovery of ribosomes at synapses indicated the potential for local protein synthesis in response to stimulation. miRNAs play a key role in this process as evidenced by their role in normal neuronal development and function and in neurological disease. miRNA production is regulated and once bound by AGO2, the ensuing RISC complex is able to bind mRNAs and direct their translation suppression and degradation. However, other RNA binding proteins, including FMRP and MOV10, regulate AGO2 association with the miRNA recognition element (MRE) in target mRNAs. AGO2 itself is regulated by post-translational modifications, and neuronal activity controls post-translational modifications of FMRP and MOV10 that lead to their regulation and degradation. In addition, RNA localization at the synapse is a critical regulated event that depends on both cis sequences in the mRNA and the identity of the bound RNA binding proteins.

scholarly journals Assembly of the α-Globin mRNA Stability Complex Reflects Binary Interaction between the Pyrimidine-Rich 3′ Untranslated Region Determinant and Poly(C) Binding Protein αCP

1999 ◽  
Vol 19 (7) ◽  
pp. 4572-4581 ◽  
Author(s):  
Alexander N. Chkheidze ◽  
Dmitry L. Lyakhov ◽  
Alexander V. Makeyev ◽  
Julia Morales ◽  
Jian Kong ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Untranslated Region ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Specific Binding ◽  
Direct Interaction ◽  
Binary Interaction ◽  
Globin Mrna ◽  
Hnrnp K

ABSTRACT Globin mRNAs accumulate to 95% of total cellular mRNA during terminal erythroid differentiation, reflecting their extraordinary stability. The stability of human α-globin mRNA is paralleled by formation of a sequence-specific RNA-protein (RNP) complex at a pyrimidine-rich site within its 3′ untranslated region (3′UTR), the α-complex. The proteins of the α-complex are widely expressed. The α-complex or a closely related complex also assembles at pyrimidine-rich 3′UTR segments of other stable mRNAs. These data suggest that the α-complex may constitute a general determinant of mRNA stability. One or more αCPs, members of a family of hnRNP K-homology domain poly(C) binding proteins, are essential constituents of the α-complex. The ability of αCPs to homodimerize and their reported association with additional RNA binding proteins such as AU-rich binding factor 1 (AUF1) and hnRNP K have suggested that the α-complex is a multisubunit structure. In the present study, we have addressed the composition of the α-complex. An RNA titration recruitment assay revealed that αCPs were quantitatively incorporated into the α-complex in the absence of associated AUF1 and hnRNP K. A high-affinity direct interaction between each of the three major αCP isoforms and the α-globin 3′UTR was detected, suggesting that each of these proteins might be sufficient for α-complex assembly. This sufficiency was further supported by the sequence-specific binding of recombinant αCPs to a spectrum of RNA targets. Finally, density sedimentation analysis demonstrated that the α-complex could accommodate only a single αCP. These data established that a single αCP molecule binds directly to the α-globin 3′UTR, resulting in a simple binary structure for the α-complex.

scholarly journals Regulation of mammalian ribonucleotide reductase R1 mRNA stability is mediated by a ribonucleotide reductase R1 mRNA 3′-untranslated region cis-trans interaction through a protein kinase C-controlled pathway

1994 ◽  
Vol 302 (1) ◽  
pp. 125-132 ◽  
Author(s):  
F Y Chen ◽  
F M Amara ◽  
J A Wright
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Okadaic Acid ◽  
Ribonucleotide Reductase ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Rna Binding ◽  
Kinase C ◽  
The Stability ◽  
Dose Dependent

Ribonucleotide reductase catalyses the reaction that eventually provides the four deoxyribonucleotides required for the synthesis and repair of DNA. U.v.-cross-linking and band-shift experiments have identified in COS 7 monkey cells an approx. 57 kDa ribonucleotide reductase R1 mRNA-binding protein called R1BP, which binds specifically to a 49-nt region of the R1 mRNA 3′-untranslated region (3′UTR). The R1BP-RNA binding activity was down-regulated by the tumour promoters phorbol 12-myristate 13-acetate (PMA; ‘TPA’) and okadaic acid, and up-regulated by the protein kinase C inhibitor staurosporine, in a dose-dependent fashion. Furthermore, staurosporine treatment decreased the stability of R1 and CAT (chloramphenicol acetyltransferase)/R1 hybrid mRNAs, whereas PMA and okadaic acid increased the stability of these messages, in a dose-dependent manner. In contrast, treatment of cells with forskolin, a protein kinase A inhibitor, did not alter either R1BP-RNA binding or R1 mRNA-stability characteristics. Transfectants containing R1 or CAT/R1 cDNA constructs with a deletion of the 49-nt 3′UTR sequence failed to respond in message-stability studies to the effects of PMA, staurosporine or okadaic acid. These observations indicate that a protein kinase C signal pathway regulates ribonucleotide reductase R1 gene expression post-transcriptionally, through a mechanism involving a specific cis-trans interaction at a 49-nt region within the R1 mRNA 3′UTR.

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