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Current Protocols in Neuroscience Jun 2019Ribosome tagging has become a very useful in vivo approach for analyzing gene expression and mRNA translation in specific cell types that are difficult and time...
Ribosome tagging has become a very useful in vivo approach for analyzing gene expression and mRNA translation in specific cell types that are difficult and time consuming to isolate by conventional methods. The approach is based on selectively expressing a hemagglutinin A (HA)-tagged ribosomal protein in a target cell type and then using antibodies against HA to purify the polysomes and associated mRNAs from the target cell. The original approach makes use of a mouse line (RiboTag) harboring a modified allele of Rpl22 (Rpl22-HA) that is induced by the action of Cre recombinase. The Rpl22-HA gene can also be introduced into the animal by stereotaxic injection of an AAV-DIO-Rpl22-HA that is then activated in Cre-expressing cells. Both methods for tagging ribosomes facilitate the immunoprecipitation of ribosome-bound mRNAs and their analysis by qRT-PCR or RNA-Seq. This protocol will discuss the technical procedures and describe important considerations relevant to the analysis of the data. © 2019 by John Wiley & Sons, Inc.
Topics: Animals; Gene Expression; Mice; Mice, Inbred C57BL; Mice, Transgenic; RNA, Messenger; Real-Time Polymerase Chain Reaction; Ribosomes; Sequence Analysis, RNA
PubMed: 31216392
DOI: 10.1002/cpns.77 -
Nucleic Acids Research Feb 2020Ribosomopathies are diseases caused by defects in ribosomal constituents or in factors with a role in ribosome assembly. Intriguingly, congenital ribosomopathies display... (Review)
Review
Ribosomopathies are diseases caused by defects in ribosomal constituents or in factors with a role in ribosome assembly. Intriguingly, congenital ribosomopathies display a paradoxical transition from early symptoms due to cellular hypo-proliferation to an elevated cancer risk later in life. Another association between ribosome defects and cancer came into view after the recent discovery of somatic mutations in ribosomal proteins and rDNA copy number changes in a variety of tumor types, giving rise to somatic ribosomopathies. Despite these clear connections between ribosome defects and cancer, the molecular mechanisms by which defects in this essential cellular machinery are oncogenic only start to emerge. In this review, the impact of ribosomal defects on the cellular function and their mechanisms of promoting oncogenesis are described. In particular, we discuss the emerging hallmarks of ribosomopathies such as the appearance of 'onco-ribosomes' that are specialized in translating oncoproteins, dysregulation of translation-independent extra-ribosomal functions of ribosomal proteins, rewired cellular protein and energy metabolism, and extensive oxidative stress leading to DNA damage. We end by integrating these findings in a model that can provide an explanation how ribosomopathies could lead to the transition from hypo- to hyper-proliferation in bone marrow failure syndromes with elevated cancer risk.
Topics: Bone Marrow Failure Disorders; Carcinogenesis; Cell Proliferation; Humans; Mitochondria; Mutation; Neoplasms; Protein Biosynthesis; RNA, Ribosomal; Ribosomal Proteins; Ribosomes
PubMed: 31350888
DOI: 10.1093/nar/gkz637 -
Cell Stem Cell Jul 2021Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains...
Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and ex vivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.
Topics: Hematopoietic Stem Cells; Protein Biosynthesis; Ribosomes
PubMed: 33711283
DOI: 10.1016/j.stem.2021.02.008 -
The Journal of Biological Chemistry 2021This essay, which was written to commemorate the 50th anniversary of the Protein Data Bank, opens with some comments about the intentions of the scientists who pressed... (Review)
Review
This essay, which was written to commemorate the 50th anniversary of the Protein Data Bank, opens with some comments about the intentions of the scientists who pressed for its establishment and the nature of services it provides. It includes a brief account of the events that resulted in the determination of the crystal structure of the large ribosomal subunit from Haloarcula marismortui. The magnitude of the challenge the first ribosome crystal structures posed for the PDB is commented upon, and in the description of subsequent developments in the ribosome structure field that follows, it is pointed out that cryo-EM has replaced X-ray crystallography as the method of choice for investigating ribosome structure.
Topics: Cryoelectron Microscopy; Crystallography, X-Ray; Databases, Protein; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Proteins; Ribosomes
PubMed: 33744288
DOI: 10.1016/j.jbc.2021.100561 -
Current Opinion in Rheumatology Jan 2022Translation of genetic information encoded within mRNA molecules by ribosomes into proteins is a key part of the central dogma of molecular biology. Despite the central... (Review)
Review
PURPOSE OF REVIEW
Translation of genetic information encoded within mRNA molecules by ribosomes into proteins is a key part of the central dogma of molecular biology. Despite the central position of the ribosome in the translation of proteins, and considering the major proteomic changes that occur in the joint during osteoarthritis development and progression, the ribosome has received very limited attention as driver of osteoarthritis pathogenesis.
RECENT FINDINGS
We provide an overview of the limited literature regarding this developing topic for the osteoarthritis field. Recent key findings that connect ribosome biogenesis and activity with osteoarthritis include: ribosomal RNA transcription, processing and maturation, ribosomal protein expression, protein translation capacity and preferential translation.
SUMMARY
The ribosome as the central cellular protein synthesis hub is largely neglected in osteoarthritis research. Findings included in this review reveal that in osteoarthritis, ribosome aberrations have been found from early-stage ribosome biogenesis, through ribosome build-up and maturation, up to preferential translation. Classically, osteoarthritis has been explained as an imbalance between joint tissue anabolism and catabolism. We postulate that osteoarthritis can be interpreted as an acquired ribosomopathy. This hypothesis fine-tunes the dogmatic anabolism/katabolism point-of-view, and may provide novel molecular opportunities for the development of osteoarthritis disease-modifying treatments.
Topics: Humans; Osteoarthritis; Proteomics; RNA, Ribosomal; Ribosomal Proteins; Ribosomes
PubMed: 34750309
DOI: 10.1097/BOR.0000000000000858 -
Chromosome Research : An International... Mar 2019Ribosomal DNA, the topic of this special issue, has long fascinated biologists. The RNA products of the ribosomal DNA are the ribosomal RNAs that are part of the...
Ribosomal DNA, the topic of this special issue, has long fascinated biologists. The RNA products of the ribosomal DNA are the ribosomal RNAs that are part of the ribosome. In this special issue, we focus on the sequence, molecular organization, repair, stability, copy number, and peculiar genetics of this region of the genome. The locus can impact not only the translational capability of cells, but also genome organization, stability and integrity, providing a link between translation and chromosome biology.
Topics: Animals; Chromosomes; DNA Replication; DNA, Ribosomal; Gene Expression Regulation; Genome; Genomics; Humans; Ribosomes
PubMed: 30663012
DOI: 10.1007/s10577-018-9601-4 -
Biochemistry Nov 2019As the influence of translation rates on protein folding and function has come to light, the mechanisms by which translation speed is modulated have become an important... (Review)
Review
As the influence of translation rates on protein folding and function has come to light, the mechanisms by which translation speed is modulated have become an important issue. One mechanism entails the generation of force by the nascent protein. Cotranslational processes, such as nascent protein folding, the emergence of unfolded nascent chain segments from the ribosome's exit tunnel, and insertion of the nascent chain into or translocation of the nascent chain through membranes, can generate forces that are transmitted back to the peptidyl transferase center and affect translation rates. In this Perspective, we examine the processes that generate these forces, the mechanisms of transmission along the ribosomal exit tunnel to the peptidyl transferase center, and the effects of force on the ribosome's catalytic cycle. We also discuss the physical models that have been developed to predict and explain force generation for individual processes and speculate about other processes that may generate forces that have yet to be tested.
Topics: Animals; Biomechanical Phenomena; Humans; Kinetics; Models, Molecular; Peptidyl Transferases; Protein Biosynthesis; Ribosomes
PubMed: 31134795
DOI: 10.1021/acs.biochem.9b00260 -
Cells Mar 2019Ribosomopathies are congenital diseases with defects in ribosome assembly and are characterized by elevated cancer risks. Additionally, somatic mutations in ribosomal... (Review)
Review
Ribosomopathies are congenital diseases with defects in ribosome assembly and are characterized by elevated cancer risks. Additionally, somatic mutations in ribosomal proteins have recently been linked to a variety of cancers. Despite a clear correlation between ribosome defects and cancer, the molecular mechanisms by which these defects promote tumorigenesis are unclear. In this review, we focus on the emerging mechanisms that link ribosomal defects in ribosomopathies to cancer progression. This includes functional "onco-specialization" of mutant ribosomes, extra-ribosomal consequences of mutations in ribosomal proteins and ribosome assembly factors, and effects of ribosomal mutations on cellular stress and metabolism. We integrate some of these recent findings in a single model that can partially explain the paradoxical transition from hypo- to hyperproliferation phenotypes, as observed in ribosomopathies. Finally, we discuss the current and potential strategies, and the associated challenges for therapeutic intervention in ribosome-mutant diseases.
Topics: Animals; Carcinogenesis; Humans; Models, Biological; Neoplasms; Organelle Biogenesis; Ribosomes
PubMed: 30862070
DOI: 10.3390/cells8030229 -
Cells Oct 2019Subverting the conventional concept of "the" ribosome, a wealth of information gleaned from recent studies is revealing a much more diverse and dynamic ribosomal reality... (Review)
Review
Subverting the conventional concept of "the" ribosome, a wealth of information gleaned from recent studies is revealing a much more diverse and dynamic ribosomal reality than has traditionally been thought possible. A diverse array of researchers is collectively illuminating a universe of heterogeneous and adaptable ribosomes harboring differences in composition and regulatory capacity: These differences enable specialization. The expanding universe of ribosomes not only comprises an incredible richness in ribosomal specialization between species, but also within the same tissues and even cells. In this review, we discuss ribosomal heterogeneity and speculate how the emerging understanding of the ribosomal repertoire is impacting the biological sciences today. Targeting pathogen-specific and pathological "diseased" ribosomes promises to provide new treatment options for patients, and potential applications for "designer ribosomes" are within reach. Our deepening understanding of and ability to manipulate the ribosome are establishing both the technological and theoretical foundations for major advances for the 21 century and beyond.
Topics: Animals; Humans; Ribosomes; Species Specificity
PubMed: 31590378
DOI: 10.3390/cells8101205 -
Acta Histochemica Apr 2017There is growing evidence indicating that the human pathological conditions characterized by an up-regulated ribosome biogenesis are at an increased risk of cancer... (Review)
Review
There is growing evidence indicating that the human pathological conditions characterized by an up-regulated ribosome biogenesis are at an increased risk of cancer onset. At the basis of this relationship is the close interconnection between the ribosome biogenesis and cell proliferation. Cell proliferation-stimulating factors also stimulate ribosome production, while the ribosome biogenesis rate controls the cell cycle progression. The major tumour suppressor, the p53 protein, plays an important balancing role between the ribosome biogenesis rate and the cell progression through the cell cycle phases. The perturbation of ribosome biogenesis stabilizes and activates p53, with a consequent cell cycle arrest and/or apoptotic cell death, whereas an up-regulated ribosome production down-regulates p53 expression and activity, thus facilitating neoplastic transformation. In the present review we describe the interconnection between ribosome biogenesis and cell proliferation, while highlighting the mechanisms by which quantitative changes in ribosome biogenesis may induce cancer.
Topics: Cell Cycle; Gene Expression Regulation, Neoplastic; Humans; Neoplasms; Ribosomes
PubMed: 28168996
DOI: 10.1016/j.acthis.2017.01.009