scholarly journals Mammalian Y RNAs are modified at discrete guanosine residues with N-glycans

2019 ◽  
Author(s):  
Ryan A. Flynn ◽  
Benjamin A. H. Smith ◽  
Alex G. Johnson ◽  
Kayvon Pedram ◽  
Benson M. George ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Cultured Cells ◽  
Mammalian Species ◽  
Cell Types ◽  
Intramolecular Interactions ◽  
Small Noncoding Rnas ◽  
Select Group ◽  
Domains Of Life

ABSTRACTGlycans modify lipids and proteins to mediate inter- and intramolecular interactions across all domains of life. RNA, another multifaceted biopolymer, is not thought to be a major target of glycosylation. Here, we challenge this view with evidence that mammalian cells use RNA as a third scaffold for glycosylation in the secretory pathway. Using a battery of chemical and biochemical approaches, we find that a select group of small noncoding RNAs including Y RNAs are modified with complex, sialylated N-glycans (glycoRNAs). These glycoRNA are present in multiple cell types and mammalian species, both in cultured cells andin vivo. Finally, we find that RNA glycosylation depends on the canonical N-glycan biosynthetic machinery within the ER/Golgi luminal spaces. Collectively, these findings suggest the existence of a ubiquitous interface of RNA biology and glycobiology suggesting an expanded role for glycosylation beyond canonical lipid and protein scaffolds.

scholarly journals Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2148-2154 ◽  
Author(s):  
R D McKinnon ◽  
P Danielson ◽  
M A Brow ◽  
F E Bloom ◽  
J G Sutcliffe
Keyword(s):  
Rat Brain ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Culture Conditions ◽  
Specific Expression ◽  
Peripheral Tissues ◽  
Primate Cell

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

Expression of small cytoplasmic transcripts of the rat identifier element in vivo and in cultured cells

1987 ◽  
Vol 7 (6) ◽  
pp. 2148-2154
Author(s):  
R D McKinnon ◽  
P Danielson ◽  
M A Brow ◽  
F E Bloom ◽  
J G Sutcliffe
Keyword(s):  
Rat Brain ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Culture Conditions ◽  
Specific Expression ◽  
Peripheral Tissues ◽  
Primate Cell

We examined the level of expression of small RNA transcripts hybridizing to a rodent repetitive DNA element, the identifier (ID) sequence, in a variety of cell types in vivo and in cultured mammalian cells. A 160-nucleotide (160n) cytoplasmic poly(A)+ RNA (BC1) appeared in late embryonic and early postnatal rat brain development, was enriched in the cerebral cortex, and appeared to be restricted to neural tissue and the anterior pituitary gland. A 110n RNA (BC2) was specifically enriched in brain, especially the postnatal cortex, but was detectable at low levels in peripheral tissues. A third, related 75n poly(A)- RNA (T3) was found in rat brain and at lower levels in peripheral tissues but was very abundant in the testes. The BC RNAs were found in a variety of rat cell lines, and their level of expression was dependent upon cell culture conditions. A rat ID probe detected BC-like RNAs in mouse brain but not liver and detected a 200n RNA in monkey brain but not liver at lower hybridization stringencies. These RNAs were expressed by mouse and primate cell lines. Thus, tissue-specific expression of small ID-sequence-related transcripts is conserved among mammals, but the tight regulation found in vivo is lost by cells in culture.

Quantitative Studies of Fibronectin Adsorption on Submicron Scaffolds

2012 ◽  
Author(s):  
Shawn Regis ◽  
Manisha Jassal ◽  
Sina Youssefian ◽  
Nima Rahbar ◽  
Sankha Bhowmick
Keyword(s):  
Surface Functionalization ◽  
Cultured Cells ◽  
Cell Attachment ◽  
Md Simulations ◽  
Cell Types ◽  
Biological Processes ◽  
Integrin Receptors ◽  
Tissue Engineering Scaffolds ◽  
Quantitative Studies

Fibronectin plays a crucial role in adhesion of several cell types, mainly due to the fact that it is recognized by at least ten different integrin receptors. Since most cell types can bind to fibronectin, it becomes involved in many various biological processes. The interaction of cells with ECM proteins such as fibronectin provides the signals affecting morphology, motility, gene expression, and survival of cells [1]. Fibronectin exists in both soluble and insoluble forms; soluble fibronectin is secreted by cells and exits in cell media or body fluids, whereas insoluble fibronectin exists in tissues or the extracellular matrix of cultured cells [2]. The ability to control adsorption of fibronectin on tissue engineering scaffolds would therefore play a huge role in controlling cell attachment and survival in vivo. This can be achieved through surface functionalization of the scaffolds. The goal of these studies is to use molecular dynamics (MD) simulations to mechanistically understand how fibronectin adsorption is enhanced by surface functionalization of submicron scaffolds.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Vol 116 (1) ◽  
pp. 303-312 ◽  
Author(s):  
Erol C. Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

scholarly journals MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo

2018 ◽  
Author(s):  
Erol Can Bayraktar ◽  
Lou Baudrier ◽  
Ceren Özerdem ◽  
Caroline A. Lewis ◽  
Sze Ham Chan ◽  
...  
Keyword(s):  
Metabolite Profiling ◽  
Cultured Cells ◽  
Transgenic Animals ◽  
Cell Types ◽  
Tissue Level ◽  
Specific Cell ◽  
Mammalian Tissues ◽  
Cell Type Specific ◽  
Untargeted Metabolite Profiling

ABSTRACTMitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell-types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially-localized 3XHA epitope-tag (“MITO-Tag”) for the fast isolation of mitochondria from cultured cells to now generate “MITO-Tag Mice.” Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology and our strategy should be generally applicable for studying other mammalian organelles in specific cell-types in vivo.

scholarly journals Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer

2003 ◽  
Vol 2003 (2) ◽  
pp. 79-91 ◽  
Author(s):  
Lindsay J. Stanbridge ◽  
Vincent Dussupt ◽  
Norman J. Maitland
Keyword(s):  
Prostate Cancer ◽  
Gene Therapy ◽  
Viral Entry ◽  
Mammalian Cells ◽  
Primary Tumour ◽  
Genetic Material ◽  
Cell Types ◽  
Attractive Alternative

Current curative strategies for prostate cancer are restricted to the primary tumour, and the effect of treatments to control metastatic disease is not sustained. Therefore, the application of gene therapy to prostate cancer is an attractive alternative. Baculoviruses are highly restricted insect viruses, which can enter, but not replicate in mammalian cells. Baculoviruses can incorporate large amounts of extra genetic material, and will express transgenes in mammalian cells when under the control of a mammalian or strong viral promoter. Successful gene delivery has been achieved both in vitro and in vivo and into both dividing and nondividing cells, which is important since prostate cancers divide relatively slowly. In addition, the envelope protein gp64 is sufficiently mutable to allow targeted transduction of particular cell types. In this review, the advantages of using baculoviruses for prostate cancer gene therapy are explored, and the mechanisms of viral entry and transgene expression are described.

scholarly journals Binding of Kingella kingae RtxA Toxin Depends on Cell Surface Oligosaccharides, but Not on β2 Integrins

2020 ◽  
Vol 21 (23) ◽  
pp. 9092
Author(s):  
Waheed Ur Rahman ◽  
Adriana Osickova ◽  
Nela Klimova ◽  
Jinery Lora ◽  
Nataliya Balashova ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mammalian Cells ◽  
Mammalian Species ◽  
Cell Types ◽  
Surface Structures ◽  
Target Cells ◽  
Kingella Kingae ◽  
Toxin Binding ◽  
Cell Surface Structures ◽  
Β2 Integrins

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the β2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the β2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted β2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.

scholarly journals Achieving tight control of a photoactivatable Cre recombinase gene switch: new design strategies and functional characterization in mammalian cells and rodent

2019 ◽  
Vol 47 (17) ◽  
pp. e97-e97 ◽  
Author(s):  
Kyle Meador ◽  
Christina L Wysoczynski ◽  
Aaron J Norris ◽  
Jason Aoto ◽  
Michael R Bruchas ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Functional Characterization ◽  
Cre Recombinase ◽  
Common Mechanism ◽  
Protein Fragments ◽  
Low Background ◽  
Wide Range ◽  
Split Protein

AbstractA common mechanism for inducibly controlling protein function relies on reconstitution of split protein fragments using chemical or light-induced dimerization domains. A protein is split into fragments that are inactive on their own, but can be reconstituted after dimerization. As many split proteins retain affinity for their complementary half, maintaining low activity in the absence of an inducer remains a challenge. Here, we systematically explore methods to achieve tight regulation of inducible proteins that are effective despite variation in protein expression level. We characterize a previously developed split Cre recombinase (PA-Cre2.0) that is reconstituted upon light-induced CRY2-CIB1 dimerization, in cultured cells and in vivo in rodent brain. In culture, PA-Cre2.0 shows low background and high induced activity over a wide range of expression levels, while in vivo the system also shows low background and sensitive response to brief light inputs. The consistent activity stems from fragment compartmentalization that shifts localization toward the cytosol. Extending this work, we exploit nuclear compartmentalization to generate light-and-chemical regulated versions of Cre recombinase. This work demonstrates in vivo functionality of PA-Cre2.0, describes new approaches to achieve tight inducible control of Cre DNA recombinase, and provides general guidelines for further engineering and application of split protein fragments.

scholarly journals Rapid Maturation of Glycoprotein Hormone Free α-Subunit (GPHα) and GPHαα Homodimers

2007 ◽  
Vol 21 (10) ◽  
pp. 2551-2564 ◽  
Author(s):  
Jean-Michel Krause ◽  
Peter Berger ◽  
Jordi Roig ◽  
Vinod Singh ◽  
Wolfgang E. Merz
Keyword(s):  
Hela Cells ◽  
Secretory Pathway ◽  
Cell Types ◽  
Subunit Interaction ◽  
Glycoprotein Hormone ◽  
Α Subunit ◽  
Self Association ◽  
Choriocarcinoma Cells ◽  
Loop 2

Abstract The dynamics of glycoprotein hormone α-subunit (GPHα) maturation and GPHαα homodimer formation were studied in presence (JEG-3 choriocarcinoma cells) and absence (HeLa cells) of hCGβ. In both cases, the major initially occurring GPHα variant in [35S]Met/Cys-labeled cells carried two N-glycans (Mr app = 22 kDa). Moreover, a mono-N-glycosylated in vivo association-incompetent GPHα variant (Mr app = 18 kDa) was observed. In JEG-3 cells the early 22-kDa GPHα either associated with hCGβ, or showed self-association to yield GPHαα homodimers, or was later converted into heavily glycosylated large free GPHα (Mr app = 24 kDa). Micro-preparative isolation of intracellular GPHαα homodimers of JEG-3 cells and their conversion by reduction revealed that they consisted of 22-kDa GPHα monomers and not of large free GPHα. In HeLa cells, the large free GPHα variant was not observed, whereas GPHαα homodimers were present. Intracellularly, early GPHαα homodimers (35 kDa) and late variants (JEG-3: 44 kDa, HeLa: 39 kDa) were found. Both cell types secreted 45 kDa GPHαα homodimers. Large free GPHα and GPHαα homodimers were more rapidly sialylated than hCG αβ-heterodimers indicating a sequestration mechanism in the secretory pathway. In GPHαα homo- as well as hCG αβ-heterodimers the subunit interaction site, located on loop 2 of GPHα (amino acids 33–42), became immunologically inaccessible indicating similar spatial orientation of GPHα in both types of dimers. The studies demonstrate the formation, in vivo dynamics of GPHαα homodimers, and the pathways of the cellular metabolism of variants of GPHα, monoglycosylated GPHα and large free GPHα.

scholarly journals The multiple facets of the Golgi reassembly stacking proteins

2011 ◽  
Vol 433 (3) ◽  
pp. 423-433 ◽  
Author(s):  
Fabian P. Vinke ◽  
Adam G. Grieve ◽  
Catherine Rabouille
Keyword(s):  
Mammalian Cells ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Mitotic Checkpoint ◽  
Biochemical Properties ◽  
C Terminus ◽  
Unconventional Secretion ◽  
Golgi Ribbon

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.

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