scholarly journals Neuronal ribosomes exhibit dynamic and context-dependent exchange of ribosomal proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Claudia M. Fusco ◽  
Kristina Desch ◽  
Aline R. Dörrbaum ◽  
Mantian Wang ◽  
Anja Staab ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosome Profiling ◽  
Metabolic Labeling ◽  
Local Protein Synthesis ◽  
Translational Machinery ◽  
Neuronal Synapses ◽  
Cellular Environment ◽  
Neuronal Processes ◽  
Local Protein

AbstractOwing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation was independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis.

scholarly journals Neuronal ribosomes dynamically exchange ribosomal proteins in a context-dependent manner.

2021 ◽  
Author(s):  
Claudia M. Fusco ◽  
Kristina Desch ◽  
Aline R. Doerrbaum ◽  
Mantian Wang ◽  
Anja Staab ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosome Profiling ◽  
Metabolic Labeling ◽  
Dependent Manner ◽  
Dense Network ◽  
Translational Machinery ◽  
Neuronal Synapses ◽  
Cellular Environment ◽  
Neuronal Processes

Owing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs quickly associated with translating ribosomes in the cytoplasm and that this incorporation is independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (in response to oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing remote neuronal synapses a rapid solution to the relatively slow and energy-demanding requirement of nuclear ribosome biogenesis.

scholarly journals Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments

2018 ◽  
Author(s):  
Anne-Sophie Hafner ◽  
Paul G. Donlin-Asp ◽  
Beulah Leitch ◽  
Etienne Herzog ◽  
Erin M. Schuman
Keyword(s):  
Protein Synthesis ◽  
Mouse Brain ◽  
Brain Slices ◽  
Metabolic Labeling ◽  
Presynaptic Terminals ◽  
Ample Evidence ◽  
Local Protein Synthesis ◽  
Axon Terminals ◽  
Neuronal Dendrites ◽  
Local Protein

AbstractThere is ample evidence for localized mRNAs and protein synthesis in neuronal dendrites, however, demonstrations of these processes in presynaptic terminals are limited. We used expansion microscopy to resolve pre- and postsynaptic compartments in brain slices. Most presynaptic terminals in the hippocampus and forebrain contained mRNA and ribosomes. We sorted fluorescently labeled synaptosomes from mouse brain and then sequenced hundreds of mRNA species present within excitatory boutons. After brief metabolic labeling, more them 30% of all presynaptic terminals exhibited a signal, providing evidence for ongoing protein synthesis. We tested different classic plasticity paradigms and observed unique patterns of rapid pre- and/or postsynaptic translation. Thus presynaptic terminals are translationally competent and local protein synthesis is differentially recruited to drive compartment-specific phenotypes that underlie different forms of plasticity.One sentence summaryProtein synthesis occurs in all synaptic compartments, including excitatory and inhibitory axon terminals.

scholarly journals Nonphosphorylated Human La Antigen Interacts with Nucleolin at Nucleolar Sites Involved in rRNA Biogenesis

2004 ◽  
Vol 24 (24) ◽  
pp. 10894-10904 ◽  
Author(s):  
Robert V. Intine ◽  
Miroslav Dundr ◽  
Alex Vassilev ◽  
Elena Schwartz ◽  
Yingmin Zhao ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Affinity Purification ◽  
Close Association ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Rna Recognition Motif ◽  
Translational Machinery ◽  
Terminal Domain

ABSTRACT La is a RNA-binding protein implicated in multiple pathways related to the production of tRNAs, ribosomal proteins, and other components of the translational machinery (D. J. Kenan and J. D. Keene, Nat. Struct. Mol. Biol. 11 :303-305, 2004). While most La is phosphorylated and resides in the nucleoplasm, a fraction is in the nucleolus, the site of ribosome production, although the determinants of this localization are incompletely known. In addition to its conserved N-terminal domain, human La harbors a C-terminal domain that contains an atypical RNA recognition motif and a short basic motif (SBM) adjacent to phosphoserine-366. We report that nonphosphorylated La (npLa) is concentrated in nucleolar sites that correspond to the dense fibrillar component that harbors nascent pol I transcripts as well as fibrillarin and nucleolin, which function in early phases of rRNA maturation. Affinity purification and native immunoprecipitation of La and fluorescence resonance energy transfer in the nucleolus reveal close association with nucleolin. Moreover, La lacking the SBM does not localize to nucleoli. Lastly, La exhibits SBM-dependent, phosphorylation-sensitive interaction with nucleolin in a yeast two-hybrid assay. The data suggest that interaction with nucleolin is, at least in part, responsible for nucleolar accumulation of La and that npLa may be involved in ribosome biogenesis.

scholarly journals FMRP links optimal codons to mRNA stability in neurons

2020 ◽  
Vol 117 (48) ◽  
pp. 30400-30411
Author(s):  
Huan Shu ◽  
Elisa Donnard ◽  
Botao Liu ◽  
Suna Jung ◽  
Ruijia Wang ◽  
...  
Keyword(s):  
Mrna Stability ◽  
Cortical Neurons ◽  
Rna Binding ◽  
Fragile X ◽  
Transcript Abundance ◽  
Ribosome Profiling ◽  
Metabolic Labeling ◽  
Genetic Rescue ◽  
Translational Machinery ◽  
Optimal Codons

Fragile X syndrome (FXS) is caused by inactivation of theFMR1gene and loss of encoded FMRP, an RNA binding protein that represses translation of some of its target transcripts. Here we use ribosome profiling and RNA sequencing to investigate the dysregulation of translation in the mouse brain cortex. We find that most changes in ribosome occupancy on hundreds of mRNAs are largely driven by dysregulation in transcript abundance. Many down-regulated mRNAs, which are mostly responsible for neuronal and synaptic functions, are highly enriched for FMRP binding targets. RNA metabolic labeling demonstrates that, in FMRP-deficient cortical neurons, mRNA down-regulation is caused by elevated degradation and is correlated with codon optimality. Moreover, FMRP preferentially binds mRNAs with optimal codons, suggesting that it stabilizes such transcripts through direct interactions via the translational machinery. Finally, we show that the paradigm of genetic rescue of FXS-like phenotypes in FMRP-deficient mice by deletion of theCpeb1gene is mediated by restoration of steady-state RNA levels and consequent rebalancing of translational homeostasis. Our data establish an essential role of FMRP in codon optimality-dependent mRNA stability as an important factor in FXS.

scholarly journals O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Core Ribosomal Proteins

2010 ◽  
Vol 21 (12) ◽  
pp. 1922-1936 ◽  
Author(s):  
Quira Zeidan ◽  
Zihao Wang ◽  
Antonio De Maio ◽  
Gerald W. Hart
Keyword(s):  
Protein Synthesis ◽  
Posttranslational Modifications ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Mammalian Target Of Rapamycin ◽  
Nuclear Staining ◽  
Mtor Signaling Pathway ◽  
Translational Machinery ◽  
Translation Factors ◽  
Nucleolar Stress

Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.

scholarly journals Monosomes actively translate synaptic mRNAs in neuronal processes

Science ◽  
2020 ◽  
Vol 367 (6477) ◽  
pp. eaay4991 ◽  
Author(s):  
Anne Biever ◽  
Caspar Glock ◽  
Georgi Tushev ◽  
Elena Ciirdaeva ◽  
Tamas Dalmay ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Active Sites ◽  
Synaptic Proteins ◽  
Messenger Rnas ◽  
Local Protein Synthesis ◽  
Relative Paucity ◽  
Polysome Profiling ◽  
Neuronal Processes ◽  
Ribosome Footprinting ◽  
Local Protein

To accommodate their complex morphology, neurons localize messenger RNAs (mRNAs) and ribosomes near synapses to produce proteins locally. However, a relative paucity of polysomes (considered the active sites of translation) detected in electron micrographs of neuronal processes has suggested a limited capacity for local protein synthesis. In this study, we used polysome profiling together with ribosome footprinting of microdissected rodent synaptic regions to reveal a surprisingly high number of dendritic and/or axonal transcripts preferentially associated with monosomes (single ribosomes). Furthermore, the neuronal monosomes were in the process of active protein synthesis. Most mRNAs showed a similar translational status in the cell bodies and neurites, but some transcripts exhibited differential ribosome occupancy in the compartments. Monosome-preferring transcripts often encoded high-abundance synaptic proteins. Thus, monosome translation contributes to the local neuronal proteome.

scholarly journals FMRP Links Optimal Codons to mRNA stability in Neurons

2019 ◽  
Author(s):  
Huan Shu ◽  
Elisa Donnard ◽  
Botao Liu ◽  
Ruijia Wang ◽  
Joel D. Richter
Keyword(s):  
Mrna Stability ◽  
Cortical Neurons ◽  
Rna Binding ◽  
Fragile X ◽  
Transcript Abundance ◽  
Ribosome Profiling ◽  
Metabolic Labeling ◽  
Genetic Rescue ◽  
Translational Machinery ◽  
Optimal Codons

AbstractFragile X syndrome (FXS) is caused by inactivation of the FMR1 gene and loss of encoded FMRP, an RNA binding protein that represses translation of some of its target transcripts. Here we use ribosome profiling and RNA-seq to investigate the dysregulation of translation in the mouse brain cortex. We find that most changes in ribosome occupancy on hundreds of mRNAs are largely driven by dysregulation in transcript abundance. Many downregulated mRNAs, which are mostly responsible for neuronal and synaptic functions, are highly enriched for FMRP binding targets. RNA metabolic labeling demonstrates that in FMRP-deficient cortical neurons, mRNA downregulation is caused by elevated degradation, and is correlated with codon optimality. Moreover, FMRP preferentially binds mRNAs with optimal codons, suggesting that it stabilizes such transcripts through direct interactions via the translational machinery. Finally, we show that the paradigm of genetic rescue of FXS-like phenotypes in FMRP-deficient mice by deletion of the Cpeb1 gene is mediated by restoration of steady state RNA levels and consequent rebalancing of translational homeostasis. Our data establish an essential role of FMRP in codon optimality-dependent mRNA stability as an important factor in FXS.

scholarly journals Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments

Science ◽  
2019 ◽  
Vol 364 (6441) ◽  
pp. eaau3644 ◽  
Author(s):  
Anne-Sophie Hafner ◽  
Paul G. Donlin-Asp ◽  
Beulah Leitch ◽  
Etienne Herzog ◽  
Erin M. Schuman
Keyword(s):  
Protein Synthesis ◽  
Mouse Brain ◽  
Metabolic Labeling ◽  
Messenger Rnas ◽  
Presynaptic Terminals ◽  
Ample Evidence ◽  
Local Protein Synthesis ◽  
Neuronal Dendrites ◽  
Brain Synaptosomes ◽  
Local Protein

There is ample evidence for localization of messenger RNAs (mRNAs) and protein synthesis in neuronal dendrites; however, demonstrations of these processes in presynaptic terminals are limited. We used expansion microscopy to resolve pre- and postsynaptic compartments in rodent neurons. Most presynaptic terminals in the hippocampus and forebrain contained mRNA and ribosomes. We sorted fluorescently labeled mouse brain synaptosomes and then sequenced hundreds of mRNA species present within excitatory boutons. After brief metabolic labeling, >30% of all presynaptic terminals exhibited a signal, providing evidence for ongoing protein synthesis. We tested different classic plasticity paradigms and observed distinct patterns of rapid pre- and/or postsynaptic translation. Thus, presynaptic terminals are translationally competent, and local protein synthesis is differentially recruited to drive compartment-specific phenotypes that underlie different forms of plasticity.

scholarly journals Monosomes actively translate synaptic mRNAs in neuronal processes

2019 ◽  
Author(s):  
Anne Biever ◽  
Caspar Glock ◽  
Georgi Tushev ◽  
Elena Ciirdaeva ◽  
Julian D. Langer ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Active Sites ◽  
Synaptic Proteins ◽  
Local Protein Synthesis ◽  
Relative Paucity ◽  
Large Size ◽  
Polysome Profiling ◽  
Neuronal Processes ◽  
Ribosome Footprinting ◽  
Local Protein

AbstractIn order to deal with their huge volume and complex morphology, neurons localize mRNAs and ribosomes near synapses to produce proteins locally. A relative paucity of polyribosomes (considered the active sites of translation) detected in electron micrographs of neuronal processes (axons and dendrites), however, has suggested a rather limited capacity for local protein synthesis. Polysome profiling together with ribosome footprinting of microdissected synaptic regions revealed that a surprisingly high number of dendritic and/or axonal transcripts were predominantly associated with monosomes (single ribosomes). Contrary to prevailing views, the neuronal monosomes were in the process of active protein synthesis (e.g. they exhibited elongation). Most mRNAs showed a similar translational status in both compartments, but some transcripts exhibited differential ribosome occupancy in the somata and neuropil. Strikingly, monosome-preferred transcripts often encoded high-abundance synaptic proteins. This work suggests a significant contribution of monosome translation to the maintenance of the local neuronal proteome. This mode of translation can presumably solve some of restricted space issues (given the large size of polysomes) and also increase the diversity of proteins made from a limited number of ribosomes available in dendrites and axons.

The in vivo functions of ARPF2 and ARRS1 in ribosomal RNA processing and ribosome biogenesis in Arabidopsis

2020 ◽  
Vol 71 (9) ◽  
pp. 2596-2611
Author(s):  
Ilyeong Choi ◽  
Young Jeon ◽  
Youngki Yoo ◽  
Hyun-Soo Cho ◽  
Hyun-Sook Pai
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Ribosome Profiling ◽  
Production Factor ◽  
Rrna Synthesis ◽  
Binary Complex ◽  
Rrna Processing ◽  
Loss Of Function

Abstract Yeast Rpf2 plays a critical role in the incorporation of 5S rRNA into pre-ribosomes by forming a binary complex with Rrs1. The protein characteristics and overexpression phenotypes of Arabidopsis Ribosome Production Factor 2 (ARPF2) and Arabidopsis Regulator of Ribosome Synthesis 1 (ARRS1) have been previously studied. Here, we analyze loss-of-function phenotypes of ARPF2 and ARRS1 using virus-induced gene silencing to determine their functions in pre-rRNA processing and ribosome biogenesis. ARPF2 silencing in Arabidopsis led to pleiotropic developmental defects. RNA gel blot analysis and circular reverse transcription–PCR revealed that ARPF2 depletion delayed pre-rRNA processing, resulting in the accumulation of multiple processing intermediates. ARPF2 fractionated primarily with the 60S ribosomal subunit. Metabolic rRNA labeling and ribosome profiling suggested that ARPF2 deficiency mainly affected 25S rRNA synthesis and 60S ribosome biogenesis. ARPF2 and ARRS1 formed the complex that interacted with the 60S ribosomal proteins RPL5 and RPL11. ARRS1 silencing resulted in growth defects, accumulation of processing intermediates, and ribosome profiling similar to those of ARPF2-silenced plants. Moreover, depletion of ARPF2 and ARRS1 caused nucleolar stress. ARPF2-deficient plants excessively accumulated anthocyanin and reactive oxygen species. Collectively, these results suggest that the ARPF2–ARRS1 complex plays a crucial role in plant growth and development by modulating ribosome biogenesis.

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