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 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 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 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 SuhB Associates with Nus Factors To Facilitate 30S Ribosome Biogenesis in Escherichia coli

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Navjot Singh ◽  
Mikhail Bubunenko ◽  
Carol Smith ◽  
David M. Abbott ◽  
Anne M. Stringer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Transcription Termination ◽  
Main Function ◽  
Dependent Manner ◽  
Reporter System ◽  
Major Function ◽  
Rrna Maturation

ABSTRACT A complex of highly conserved proteins consisting of NusB, NusE, NusA, and NusG is required for robust expression of rRNA in Escherichia coli . This complex is proposed to prevent Rho-dependent transcription termination by a process known as “antitermination.” The mechanism of this antitermination in rRNA is poorly understood but requires association of NusB and NusE with a specific RNA sequence in rRNA known as BoxA. Here, we identify a novel member of the rRNA antitermination machinery: the inositol monophosphatase SuhB. We show that SuhB associates with elongating RNA polymerase (RNAP) at rRNA in a NusB-dependent manner. Although we show that SuhB is required for BoxA-mediated antitermination in a reporter system, our data indicate that the major function of the NusB/E/A/G/SuhB complex is not to prevent Rho-dependent termination of rRNA but rather to promote correct rRNA maturation. This occurs through formation of a SuhB-mediated loop between NusB/E/BoxA and RNAP/NusA/G. Thus, we have reassigned the function of these proteins at rRNA and identified another key player in this complex. IMPORTANCE As RNA polymerase transcribes the rRNA operons in E. coli , it complexes with a set of proteins called Nus that confer enhanced rates of transcription elongation, correct folding of rRNA, and rRNA assembly with ribosomal proteins to generate a fully functional ribosome. Four Nus proteins were previously known, NusA, NusB, NusE, and NusG; here, we discover and describe a fifth, SuhB, that is an essential component of this complex. We demonstrate that the main function of this SuhB-containing complex is not to prevent premature transcription termination within the rRNA operon, as had been long claimed, but to enable rRNA maturation and a functional ribosome fully competent for translation.

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.

scholarly journals The Yeast GTPase Mtg2p Is Required for Mitochondrial Translation and Partially Suppresses an rRNA Methyltransferase Mutant,mrm2

2005 ◽  
Vol 16 (2) ◽  
pp. 954-963 ◽  
Author(s):  
Kaustuv Datta ◽  
Jennifer L. Fuentes ◽  
Janine R. Maddock
Keyword(s):  
Mitochondrial Dna ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Mitochondrial Protein ◽  
Ribosomal Subunit ◽  
Temporal Association ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Mitochondrial Translation ◽  
Mitochondrial Ribosome

The assembly of ribosomes involves the coordinated processing and modification of rRNAs with the temporal association of ribosomal proteins. This process is regulated by assembly factors such as helicases, modifying enzymes, and GTPases. In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes. In this study, we demonstrate that the Saccharomyces cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function. Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells. In addition, cells expressing a temperature-sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits. Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant, mrm2. We propose that Mtg2p is involved in mitochondrial ribosome biogenesis. Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner.

scholarly journals Upregulation of 5′-terminal oligopyrimidine mRNA translation upon loss of the ARF tumor suppressor

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kyle A. Cottrell ◽  
Ryan C. Chiou ◽  
Jason D. Weber
Keyword(s):  
Protein Synthesis ◽  
Tumor Cells ◽  
Ribosomal Proteins ◽  
Human Cancer ◽  
Mrna Translation ◽  
Ribosome Profiling ◽  
Translational Efficiency ◽  
Gene Encoding ◽  
Terminal Oligopyrimidine ◽  
Ribosome Export

AbstractTumor cells require nominal increases in protein synthesis in order to maintain high proliferation rates. As such, tumor cells must acquire enhanced ribosome production. How the numerous mutations in tumor cells ultimately achieve this aberrant production is largely unknown. The gene encoding ARF is the most commonly deleted gene in human cancer. ARF plays a significant role in regulating ribosomal RNA synthesis and processing, ribosome export into the cytoplasm, and global protein synthesis. Utilizing ribosome profiling, we show that ARF is a major suppressor of 5′-terminal oligopyrimidine mRNA translation. Genes with increased translational efficiency following loss of ARF include many ribosomal proteins and translation factors. Knockout of p53 largely phenocopies ARF loss, with increased protein synthesis and expression of 5′-TOP encoded proteins. The 5′-TOP regulators eIF4G1 and LARP1 are upregulated in Arf- and p53-null cells.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

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