scholarly journals Primer extension coupled with fragment analysis for rapid and quantitative evaluation of 5.8S rRNA isoforms

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261476
Author(s):  
Giulia Venturi ◽  
Federico Zacchini ◽  
Cinzia Lucia Vaccari ◽  
Davide Trerè ◽  
Lorenzo Montanaro
Keyword(s):  
Relative Abundance ◽  
Cell Biology ◽  
Ribosome Biogenesis ◽  
Human Cells ◽  
Primer Extension ◽  
Protective Measures ◽  
Fragment Analysis ◽  
5.8S Rrna ◽  
Human Pathology ◽  
Reverse Primer

The ribosomal RNA 5.8S is one of the four rRNAs that constitute ribosomes. In human cells, like in all eukaryotes, it derives from the extensive processing of a long precursor containing the sequence of 18S, 5.8S and 28S rRNAs. It has been confirmed also in human cells the presence of three isoforms of 5.8S rRNA: one more abundant called 5.8S short, one called 5.8S long bearing 5 extra-nucleotides at its 5’ end and one 10 nucleotide shorter called 5.8S cropped. So far, little is known about 5.8S long specific role in cell biology and its function in human pathology. The lack of studies on the three 5.8S isoforms could be due to the techniques usually applied to study ribosome biogenesis, such as Northern blot with radioactively labelled probes, that require strict protective measures, and abundant and high-quality samples. To overcome this issue, we optimized a method that combines primer extension with a fluorescently labeled reverse primer designed on the 3’ of 5.8S rRNA sequence and fragment analysis. The resulting electropherogram shows the peaks corresponding to the three isoforms of 5.8S rRNA. The estimation of the area underneath the peaks allows to directly quantify the isoforms and to express their relative abundance. The relative abundance of 5.8S long and 5.8S short remains constant using scalar dilution of RNA and in samples subjected to partial degradation. 5.8S cropped abundance varies significantly in lower concentrate RNA samples. This method allows to analyze rapidly and safely the abundance of 5.8S rRNA isoforms in samples that have been so far considered not suitable such as poorly concentrated samples, RNA derived from frozen tissue or unique samples.

Concepts of Disease: A Textbook of Human Pathology, edited by Joel C. Brunson, M.D. and Edward A. Gall, M.D. New York/London/Toronto: The Macmillan Company, 1971, 1134 pp., $23.95

PEDIATRICS ◽  
1972 ◽  
Vol 49 (3) ◽  
pp. 484-484
Author(s):  
Gordon W. Vawter
Keyword(s):  
New York ◽  
Cell Biology ◽  
Organ Systems ◽  
Human Pathology ◽  
Recent Developments ◽  
Concepts Of Disease ◽  
Novel Design

This is a lusty 7½ pound newborn book with most organ systems more than adequately mature to assure a long and fruitful life. The two senior obstetricians enlisted the aid and advice of 36 other especially knowledgeable specialists to assure successful delivery. Concepts of Disease is a textbook of pathology with a novel design, emphasizing pathology as process, and incorporates much of the newer information derived from cell biology, and from the recent developments in ultrastructural and immunofluorescent techniques.

scholarly journals Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics

2017 ◽  
Author(s):  
Gemma Hardman ◽  
Simon Perkins ◽  
Zheng Ruan ◽  
Natarajan Kannan ◽  
Philip Brownridge ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Phosphorylation ◽  
Anion Exchange ◽  
Human Cell ◽  
Cell Biology ◽  
Human Cells ◽  
Phosphopeptide Enrichment ◽  
Cell Extracts ◽  
Strong Anion Exchange ◽  
Acid Labile

Protein phosphorylation is a ubiquitous post-translational modification (PTM) that regulates all aspects of life. To date, investigation of human cell signalling has focussed on canonical phosphorylation of serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. However, mounting evidence suggests that phosphorylation of histidine also plays a central role in regulating cell biology. Phosphoproteomics workflows rely on acidic conditions for phosphopeptide enrichment, which are incompatible with the analysis of acid-labile phosphorylation such as histidine. Consequently, the extent of non-canonical phosphorylation is likely to be under-estimated. We report an Unbiased Phosphopeptide enrichment strategy based on Strong Anion Exchange (SAX) chromatography (UPAX), which permits enrichment of acid-labile phosphopeptides for characterisation by mass spectrometry. Using this approach, we identify extensive and positional phosphorylation patterns on histidine, arginine, lysine, aspartate and glutamate in human cell extracts, including 310 phosphohistidine and >1000 phospholysine sites of protein modification. Remarkably, the extent of phosphorylation on individual non-canonical residues vastly exceeds that of basal phosphotyrosine. Our study reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for exploring roles of acid-labile phosphorylation in any proteome using mass spectrometry.

scholarly journals Arabidopsis small nucleolar RNA monitors the efficient pre-rRNA processing during ribosome biogenesis

2016 ◽  
Vol 113 (42) ◽  
pp. 11967-11972 ◽  
Author(s):  
Pan Zhu ◽  
Yuqiu Wang ◽  
Nanxun Qin ◽  
Feng Wang ◽  
Jia Wang ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Higher Plants ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Developmental Defects ◽  
Protein Mutants ◽  
5.8S Rrna ◽  
Important Regulator ◽  
Nucleolar Rna

Ribosome production in eukaryotes requires the complex and precise coordination of several hundred assembly factors, including many small nucleolar RNAs (snoRNAs). However, at present, the distinct role of key snoRNAs in ribosome biogenesis remains poorly understood in higher plants. Here we report that a previously uncharacterized C (RUGAUGA)/D (CUGA) type snoRNA, HIDDEN TREASURE 2 (HID2), acts as an important regulator of ribosome biogenesis through a snoRNA–rRNA interaction. Nucleolus-localized HID2 is actively expressed in Arabidopsis proliferative tissues, whereas defects in HID2 cause a series of developmental defects reminiscent of ribosomal protein mutants. HID2 associates with the precursor 45S rRNA and promotes the efficiency and accuracy of pre-rRNA processing. Intriguingly, disrupting HID2 in Arabidopsis appears to impair the integrity of 27SB, a key pre-rRNA intermediate that generates 25S and 5.8S rRNA and is known to be vital for the synthesis of the 60S large ribosomal subunit and also produces an imbalanced ribosome profile. Finally, we demonstrate that the antisense-box of HID2 is both functionally essential and highly conserved in eukaryotes. Overall, our study reveals the vital and possibly conserved role of a snoRNA in monitoring the efficiency of pre-rRNA processing during ribosome biogenesis.

scholarly journals Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p

1999 ◽  
Vol 19 (2) ◽  
pp. 1518-1525 ◽  
Author(s):  
Nilson I. T. Zanchin ◽  
David S. Goldfarb
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Fluorescent Protein ◽  
Protein A ◽  
Nucleolar Protein ◽  
Mrna Turnover ◽  
5.8S Rrna ◽  
Multistep Process ◽  
Green Fluorescent ◽  
Two Hybrid

ABSTRACT NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001–5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3′-to-5′ exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.

Human Cloning and Stem Cells

2005 ◽  
Author(s):  
Stephen S. Hal
Keyword(s):  
Stem Cells ◽  
New York ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Human Cells ◽  
Therapeutic Cloning ◽  
Hayflick Limit ◽  
Biology Of Aging ◽  
Narrative Thread

Of the countless interviews I have conducted with scientists over the years, only once has a question prompted something of a striptease. In December of 1999, I found myself in the elegant parlor of the Union Club in New York City, chatting with a biologist named Leonard Hayflick. Although hardly a household name to the general public, Hayflick is that rare scientist whose name is permanently attached to a biological phenomenon. It is known as the “Hayflick limit,” and it derives from experiments he did in the late 19505 and early 19605 showing that human cells grown in Petri dishes will predictably replicate for a certain number of cell divisions, but then hit a wall and stop dividing. This has obvious implications for cell biology, aging, and immortality (of the in vitro sort), and indeed the Hayflick limit has been the seed around which a spirited biological debate about the biology of aging has swirled, without definitive resolution, for about four decades now. Because of this history, Hayflick has closely followed the recent work on the biology of aging and regenerative medicine, which in turn has made him a front-row spectator in the more recent controversies involving human embryonic stem cell research and “therapeutic cloning.” At the time of my conversation with Hayflick, his longtime friend Michael West was attempting to obtain human embryonic stem cells through cloning—in a particularly controversial way, by putting human cells into egg cells from ... cows. Almost as an aside, I asked Hayflick what he thought about West's experiments. Hayflick replied by rolling up his pants leg. He bared enough skin to be able to point out a tiny dimple on his right knee. “The human cells he's using for the cow work came from here,” he said. I had to stand up and lean over to see it, but there was undeniably a tiny divot in Hayflick's skin. The implications were stunning: Leonard Hayflick, the father of cellular senescence and one of the elder statesmen of gerontology, was allowing himself, in a manner of speaking, to be cloned. In addition to making the obvious point that even the most innocuous question can elicit a startling answer, Hayflick's reply offered another lesson, too: that colorful characters can provide a narrative thread for bringing a controversy to life.

scholarly journals Cohesion promotes nucleolar structure and function

2014 ◽  
Vol 25 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Bethany Harris ◽  
Tania Bose ◽  
Kenneth K. Lee ◽  
Fei Wang ◽  
Shuai Lu ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Molecular Mechanisms ◽  
Human Cells ◽  
Rna Polymerase I ◽  
Nucleolar Structure ◽  
Roberts Syndrome ◽  
Evolutionarily Conserved ◽  
Rrna Cleavage ◽  
And Function ◽  
Polymerase I

The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus.

scholarly journals Non-canonical role of wild-type SEC23B in the cellular stress response pathway

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Lamis Yehia ◽  
Darren Liu ◽  
Shuai Fu ◽  
Pranav Iyer ◽  
Charis Eng
Keyword(s):  
Stress Response ◽  
Er Stress ◽  
Cell Biology ◽  
Ribosome Biogenesis ◽  
Cellular Stress ◽  
Cellular Stress Response ◽  
Wild Type ◽  
Protein Ubiquitination ◽  
Cytoplasmic Transport ◽  
Stress Response Pathway

AbstractWhile germline recessive loss-of-function mutations in SEC23B in humans cause a rare form of anaemia, heterozygous change-of-function mutations result in increased predisposition to cancer. SEC23B encodes SEC23 homologue B, a component of coat protein complex II (COPII), which canonically transports proteins from the endoplasmic reticulum (ER) to the Golgi. Despite the association of SEC23B with anaemia and cancer, the precise pathophysiology of these phenotypic outcomes remains unknown. Recently, we reported that mutant SEC23B has non-canonical COPII-independent function, particularly within the ER stress and ribosome biogenesis pathways, and that may contribute to the pathobiology of cancer predisposition. In this study, we hypothesized that wild-type SEC23B has a baseline function within such cellular stress response pathways, with the mutant protein reflecting exaggerated effects. Here, we show that the wild-type SEC23B protein localizes to the nucleus in addition to classical distribution at the ER/Golgi interface and identify multiple putative nuclear localization and export signals regulating nuclear–cytoplasmic transport. Unexpectedly, we show that, independently of COPII, wild-type SEC23B can also localize to cell nucleoli under proteasome inhibition conditions, with distinct distribution patterns compared to mutant cells. Unbiased proteomic analyses through mass spectrometry further revealed that wild-type SEC23B interacts with a subset of nuclear proteins, in addition to central proteins in the ER stress, protein ubiquitination, and EIF2 signalling pathways. We validate the genotype-specific differential SEC23B–UBA52 (ribosomal protein RPL40) interaction. Finally, utilizing patient-derived lymphoblastoid cell lines harbouring either wild-type or mutant SEC23B, we show that SEC23B levels increase in response to ER stress, further corroborating its role as a cellular stress response sensor and/or effector. Overall, these observations suggest that SEC23B, irrespective of mutation status, has unexplored roles in the cellular stress response pathway, with implications relevant to cancer and beyond that, CDAII and normal cell biology.

scholarly journals Interactome Analysis of the Nucleocapsid Protein of SARS-CoV-2 Virus

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1155
Author(s):  
Xiaoqin Zheng ◽  
Zeyu Sun ◽  
Liang Yu ◽  
Danrong Shi ◽  
Miaojin Zhu ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Ribosome Biogenesis ◽  
Rna Binding ◽  
Affinity Purification ◽  
Human Cells ◽  
Genome Replication ◽  
N Protein ◽  
Global Pandemic ◽  
Interaction Partners ◽  
Viral Genome Replication

SARS-CoV-2 infection has caused a global pandemic that has severely damaged both public health and the economy. The nucleocapsid protein of SARS-CoV-2 is multifunctional and plays an important role in ribonucleocapsid formation and viral genome replication. In order to elucidate its functions, interaction partners of the SARS-CoV-2 N protein in human cells were identified via affinity purification and mass spectrometry. We identified 160 cellular proteins as interaction partners of the SARS-CoV-2 N protein in HEK293T and/or Calu-3 cells. Functional analysis revealed strong enrichment for ribosome biogenesis and RNA-associated processes, including ribonucleoprotein complex biogenesis, ribosomal large and small subunits biogenesis, RNA binding, catalysis, translation and transcription. Proteins related to virus defence responses, including MOV10, EIF2AK2, TRIM25, G3BP1, ZC3HAV1 and ZCCHC3 were also identified in the N protein interactome. This study comprehensively profiled the viral–host interactome of the SARS-CoV-2 N protein in human cells, and the findings provide the basis for further studies on the pathogenesis and antiviral strategies for this emerging infection.

scholarly journals An integrative proteomics method identifies a regulator of translation during stem cell maintenance and differentiation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Pierre Sabatier ◽  
Christian M. Beusch ◽  
Amir A. Saei ◽  
Mike Aoun ◽  
Noah Moruzzi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Ribosome Biogenesis ◽  
Cell Types ◽  
Parental Cell ◽  
Parental Cell Line ◽  
Web Based ◽  
The Web

AbstractDetailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and propose a method for maintaining pluripotency in vitro.

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