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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 -
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 -
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 -
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 -
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 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 -
Methods in Molecular Biology (Clifton,... 2022Protein synthesis in eukaryotes is carried out by 80S ribosomes with the help of many specific translation factors. Translation comprises four major steps: initiation,... (Review)
Review
Protein synthesis in eukaryotes is carried out by 80S ribosomes with the help of many specific translation factors. Translation comprises four major steps: initiation, elongation, termination, and ribosome recycling. In this review, we provide a comprehensive list of translation factors required for protein synthesis in yeast and higher eukaryotes and summarize the mechanisms of each individual phase of eukaryotic translation.
Topics: Protein Biosynthesis; Ribosomes; Saccharomyces cerevisiae
PubMed: 35796991
DOI: 10.1007/978-1-0716-2501-9_13 -
Biochemistry. Biokhimiia Aug 2021During protein synthesis, ribosome moves along mRNA to decode one codon after the other. Ribosome translocation is induced by a universally conserved protein, elongation... (Review)
Review
During protein synthesis, ribosome moves along mRNA to decode one codon after the other. Ribosome translocation is induced by a universally conserved protein, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. EF-G-induced translocation results in unwinding of the intramolecular secondary structures of mRNA by three base pairs at a time that renders the translating ribosome a processive helicase. Professor Alexander Sergeevich Spirin has made numerous seminal contributions to understanding the molecular mechanism of translocation. Here, we review Spirin's insights into the ribosomal translocation and recent advances in the field that stemmed from Spirin's pioneering work. We also discuss key remaining challenges in studies of translocase and helicase activities of the ribosome.
Topics: Biological Transport; Cryoelectron Microscopy; Eukaryota; Fluorescence Resonance Energy Transfer; Models, Molecular; Peptide Elongation Factor 2; Peptide Elongation Factor G; Protein Biosynthesis; RNA Helicases; RNA, Messenger; RNA, Transfer; Ribosomes; Transferases
PubMed: 34488575
DOI: 10.1134/S0006297921080095 -
Trends in Biochemical Sciences Feb 2019The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis has been speculated for over 60 years.... (Review)
Review
The existence of eukaryotic ribosomes with distinct ribosomal protein (RP) stoichiometry and regulatory roles in protein synthesis has been speculated for over 60 years. Recent advances in mass spectrometry (MS) and high-throughput analysis have begun to identify and characterize distinct ribosome stoichiometry in yeast and mammalian systems. In addition to RP stoichiometry, ribosomes host a vast array of protein modifications, effectively expanding the number of human RPs from 80 to many thousands of distinct proteoforms. Is it possible that these proteoforms combine to function as a 'ribosome code' to tune protein synthesis? We outline the specific benefits that translational regulation by specialized ribosomes can offer and discuss the means and methodologies available to correlate and characterize RP stoichiometry with function. We highlight previous research with a focus on formulating hypotheses that can guide future experiments and crack the ribosome code.
Topics: Animals; High-Throughput Screening Assays; Humans; Mass Spectrometry; Ribosomal Proteins; Ribosomes; Saccharomyces cerevisiae
PubMed: 30473427
DOI: 10.1016/j.tibs.2018.10.009 -
Wiley Interdisciplinary Reviews. RNA Jul 2021Regulation of protein synthesis is a vital step in controlling gene expression, especially during development. Over the last 10 years, it has become clear that rather... (Review)
Review
Regulation of protein synthesis is a vital step in controlling gene expression, especially during development. Over the last 10 years, it has become clear that rather than being homogeneous machines responsible for mRNA translation, ribosomes are highly heterogeneous and can play an active part in translational regulation. These "specialized ribosomes" comprise of specific protein and/or rRNA components, which are required for the translation of particular mRNAs. However, while there is extensive evidence for ribosome heterogeneity, support for specialized functions is limited. Recent work in a variety of developmental model organisms has shed some light on the biological relevance of ribosome heterogeneity. Tissue-specific expression of ribosomal components along with phenotypic analysis of ribosomal gene mutations indicate that ribosome heterogeneity and potentially specialization are common in key development processes like embryogenesis, spermatogenesis, oogenesis, body patterning, and neurogenesis. Several examples of ribosome specialization have now been proposed but strong links between ribosome heterogeneity, translation of specific mRNAs by defined mechanisms, and role of these translation events remain elusive. Furthermore, several studies have indicated that heterogeneous ribosome populations are a product of tissue-specific expression rather than specialized function and that ribosomal protein phenotypes are the result of extra-ribosomal function or overall reduced ribosome levels. Many important questions still need to be addressed in order to determine the functional importance of ribosome heterogeneity to development and disease, which is likely to vary across systems. It will be essential to dissect these issues to fully understand diseases caused by disruptions to ribosomal composition, such as ribosomopathies. This article is categorized under: Translation > Translation Regulation Translation > Ribosome Structure/Function RNA in Disease and Development > RNA in Development.
Topics: Gene Expression Regulation, Developmental; Protein Biosynthesis; RNA, Messenger; RNA, Ribosomal; Ribosomal Proteins; Ribosomes
PubMed: 33565275
DOI: 10.1002/wrna.1644