scholarly journals Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

2018 ◽  
Author(s):  
Ian M. Silverman ◽  
Sager J. Gosai ◽  
Nicholas Vrettos ◽  
Shawn W. Foley ◽  
Nathan D. Berkowitz ◽  
...  
Keyword(s):  
Mature Mirnas ◽  
Human Cells ◽  
Processing Efficiency ◽  
Stem Loop ◽  
Mature Micrornas ◽  
Mouse Cells ◽  
Low Levels ◽  
Short Hairpin Rnas ◽  
Human And Mouse

ABSTRACTMicroRNA precursors (pre-miRNAs) are short hairpin RNAs that are rapidly processed into mature microRNAs (miRNAs) in the cytoplasm. Due to their low abundance in cells, sequencing-based studies of pre-miRNAs have been limited. We successfully enriched for and deep sequenced pre-miRNAs in human cells by capturing these RNAs during their interaction with Argonaute (AGO) proteins. Using this approach, we detected > 350 pre-miRNAs in human cells and > 250 pre-miRNAs in a reanalysis of a similar study in mouse cells. We uncovered widespread trimming and non-templated additions to the 3’ ends of pre- and mature miRNAs. Additionally, we created an index for microRNA precursor processing efficiency. This analysis revealed a subset of pre-miRNAs that produce low levels of mature miRNAs despite abundant precursors, including an annotated miRNA in the 5’ UTR of the DiGeorge syndrome critical region 8 mRNA transcript. This led us to search for AGO-associated stem-loops originating from other mRNA species, which identified hundreds of putative pre-miRNAs derived from human and mouse mRNAs. In summary, we provide a wealth of information on mammalian pre-miRNAs, and identify novel microRNA and microRNA-like elements localized in mRNAs.

scholarly journals DNA methylation specifies chromosomal localization of MeCP2.

1996 ◽  
Vol 16 (1) ◽  
pp. 414-421 ◽  
Author(s):  
X Nan ◽  
P Tate ◽  
E Li ◽  
A Bird
Keyword(s):  
Dna Methylation ◽  
Cultured Cells ◽  
Centromeric Heterochromatin ◽  
Intact Protein ◽  
Chromosomal Protein ◽  
Mouse Cells ◽  
Low Levels ◽  
Mutant Cells

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

scholarly journals Novel SARS-CoV-2 encoded small RNAs in the passage to humans

2020 ◽  
Author(s):  
Gabriela A Merino ◽  
Jonathan Raad ◽  
Leandro A Bugnon ◽  
Cristian Yones ◽  
Laura Kamenetzky ◽  
...  
Keyword(s):  
Mature Mirnas ◽  
Human Cells ◽  
Species Boundaries ◽  
Rna Seq ◽  
New Host ◽  
Single Nucleotide ◽  
Stem Loop ◽  
Protein Coding ◽  
Transcriptional Reprogramming ◽  
Human Genes

Abstract Motivation The Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) has recently emerged as the responsible for the pandemic outbreak of the coronavirus disease (COVID-19). This virus is closely related to coronaviruses infecting bats and Malayan pangolins, species suspected to be an intermediate host in the passage to humans. Several genomic mutations affecting viral proteins have been identified, contributing to the understanding of the recent animal-to-human transmission. However, the capacity of SARS-CoV-2 to encode functional putative microRNAs (miRNAs) remains largely unexplored. Results We have used deep learning to discover 12 candidate stem-loop structures hidden in the viral protein-coding genome. Among the precursors, the expression of eight mature miRNAs-like sequences was confirmed in small RNA-seq data from SARS-CoV-2 infected human cells. Predicted miRNAs are likely to target a subset of human genes of which 109 are transcriptionally deregulated upon infection. Remarkably, 28 of those genes potentially targeted by SARS-CoV-2 miRNAs are down-regulated in infected human cells. Interestingly, most of them have been related to respiratory diseases and viral infection, including several afflictions previously associated with SARS-CoV-1 and SARS-CoV-2. The comparison of SARS-CoV-2 pre-miRNA sequences with those from bat and pangolin coronaviruses suggests that single nucleotide mutations could have helped its progenitors jumping inter-species boundaries, allowing the gain of novel mature miRNAs targeting human mRNAs. Our results suggest that the recent acquisition of novel miRNAs-like sequences in the SARS-CoV-2 genome may have contributed to modulate the transcriptional reprogramming of the new host upon infection.

scholarly journals A 5S rRNA/L5 complex is a precursor to ribosome assembly in mammalian cells.

1988 ◽  
Vol 106 (3) ◽  
pp. 545-556 ◽  
Author(s):  
J A Steitz ◽  
C Berg ◽  
J P Hendrick ◽  
H La Branche-Chabot ◽  
A Metspalu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
5S Rrna ◽  
5S Rna ◽  
Rna Molecules ◽  
La Protein ◽  
Rnp Complex ◽  
Mouse Cells ◽  
Protein Moiety ◽  
Human And Mouse

A novel 5S RNA-protein (RNP) complex in human and mouse cells has been analyzed using patient autoantibodies. The RNP is small (approximately 7S) and contains most of the nonribosome-associated 5S RNA molecules in HeLa cells. The 5S RNA in the particle is matured at its 3' end, consistent with the results of in vivo pulse-chase experiments which indicate that this RNP represents a later step in 5S biogenesis than a previously described 5S*/La protein complex. The protein moiety of the 5S RNP has been identified as ribosomal protein L5, which is known to be released from ribosomes in a complex with 5S after various treatments of the 60S subunit. Indirect immunofluorescence indicates that the L5/5S complex is concentrated in the nucleolus. L5 may therefore play a role in delivering 5S rRNA to the nucleolus for assembly into ribosomes.

Replication of DNA containing apurinic sites in human and mouse cells probed with parvoviruses MVM and H-1

1987 ◽  
Vol 7 (7) ◽  
pp. 2620-2624
Author(s):  
J M Vos ◽  
J Rommelaere
Keyword(s):  
Mammalian Cells ◽  
Uv Light ◽  
Host Cells ◽  
Minute Virus Of Mice ◽  
Dna Viruses ◽  
Minute Virus ◽  
Mouse Cells ◽  
Viral Mutagenesis ◽  
Human And Mouse

We studied the effect of apurinic sites on DNA replication in mouse and human cells, using parvoviruses MVM (minute virus of mice) and H-1 as probes. Although apurinic sites are efficient blocks to the replication of these single-stranded DNA viruses in vivo, depurinated parvoviruses can be reactivated if host cells have been preexposed to a subtoxic dose of UV light. The target of this conditional reactivation process is the conversion of depurinated input DNA into double-stranded replicative forms; the concomitant increase in viral mutagenesis strongly suggests that apurinic sites can be bypassed in mammalian cells.

scholarly journals p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes

2013 ◽  
Vol 201 (4) ◽  
pp. 613-629 ◽  
Author(s):  
Albert R. Davalos ◽  
Misako Kawahara ◽  
Gautam K. Malhotra ◽  
Nicholas Schaum ◽  
Jiahao Huang ◽  
...  
Keyword(s):  
Growth Arrest ◽  
High Mobility ◽  
Cytokine Secretion ◽  
Blocking Antibody ◽  
Extracellular Milieu ◽  
Hmgb1 Expression ◽  
Mouse Cells ◽  
Human And Mouse ◽  
Oncogenic Stress

Cellular senescence irreversibly arrests proliferation in response to potentially oncogenic stress. Senescent cells also secrete inflammatory cytokines such as IL-6, which promote age-associated inflammation and pathology. HMGB1 (high mobility group box 1) modulates gene expression in the nucleus, but certain immune cells secrete HMGB1 as an extracellular Alarmin to signal tissue damage. We show that nuclear HMGB1 relocalized to the extracellular milieu in senescent human and mouse cells in culture and in vivo. In contrast to cytokine secretion, HMGB1 redistribution required the p53 tumor suppressor, but not its activator ATM. Moreover, altered HMGB1 expression induced a p53-dependent senescent growth arrest. Senescent fibroblasts secreted oxidized HMGB1, which stimulated cytokine secretion through TLR-4 signaling. HMGB1 depletion, HMGB1 blocking antibody, or TLR-4 inhibition attenuated senescence-associated IL-6 secretion, and exogenous HMGB1 stimulated NF-κB activity and restored IL-6 secretion to HMGB1-depleted cells. Our findings identify senescence as a novel biological setting in which HMGB1 functions and link HMGB1 redistribution to p53 activity and senescence-associated inflammation.

scholarly journals Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Anders Lundin ◽  
Michelle J. Porritt ◽  
Himjyot Jaiswal ◽  
Frank Seeliger ◽  
Camilla Johansson ◽  
...  
Keyword(s):  
Gene Editing ◽  
Functional Integration ◽  
Cancer Drug ◽  
Cancer Drug Discovery ◽  
Mouse Cells ◽  
Genomic Editing ◽  
Human And Mouse ◽  
Mouse Genomic ◽  
In Cells

Abstract The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

scholarly journals A LIPOGENIC TOXIN RELEASED THROUGH THE INTERACTION OF A NEW CYTOPATHIC AGENT (LIPOVIRUS) AND CULTURED HUMAN CELLS

1962 ◽  
Vol 115 (5) ◽  
pp. 959-966 ◽  
Author(s):  
R. Shihman Chang ◽  
Robert P. Geyer ◽  
Stephen B. Andrus
Keyword(s):  
Gamma Globulin ◽  
Fatty Acid Content ◽  
Human Cells ◽  
Major Constituent ◽  
Rous Sarcoma ◽  
Toxic Product ◽  
Cultured Human Cells ◽  
Mouse Cells ◽  
Human Gamma Globulin ◽  
Human And Mouse

The release of a toxic product from cultured human cells infected by a new cytopathic agent (the lipovirus) was described. This toxin was dissociable from the infectious particles. It induced sudanophilia of human and mouse cells, an increase in the total fatty acid content, and a change in the major constituent fatty acids. Similar toxin was not demonstrated in cultures infected by the vaccinia herpes simplex, adeno 3, polyoma, polio 1 and 2, Coxsackie B1, parainfluenza A, and Rous sarcoma viruses. Preliminary characterization indicated that this toxin was resistant to tryptic digestion and could not be dialyzed or neutralized by human gamma globulin. It was inactivated at 58°C for 30 minutes, but stable at 37°, 4°, and –60°C. The significance of these findings is discussed.

scholarly journals Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

Blood ◽  
2011 ◽  
Vol 118 (7) ◽  
pp. 1774-1783 ◽  
Author(s):  
Jasmeen S. Merzaban ◽  
Monica M. Burdick ◽  
S. Zeineb Gadhoum ◽  
Nilesh M. Dagia ◽  
Julia T. Chu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Human Cells ◽  
Glycoprotein E ◽  
Hematopoietic Stem ◽  
Selectin Ligands ◽  
Selectin Ligand ◽  
Mouse Cells ◽  
Selectin Binding ◽  
Human And Mouse ◽  
Marrow Cells

Abstract Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin−/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved by Western blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand-1 glycoform “CLA,” and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform “HCELL.” E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL's contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures.

scholarly journals The POU Transcription Factor Oct-1 Represses Virus-Induced Interferon A Gene Expression

2005 ◽  
Vol 25 (19) ◽  
pp. 8717-8731 ◽  
Author(s):  
Thibault Mesplède ◽  
Marie-Laure Island ◽  
Nicolas Christeff ◽  
Fahrettin Petek ◽  
Janine Doly ◽  
...  
Keyword(s):  
Gene Expression ◽  
Viral Infection ◽  
Regulatory Element ◽  
Adaptive Immune ◽  
Mouse Cells ◽  
Immune System Regulation ◽  
Human And Mouse ◽  
Control Of Expression

ABSTRACT Alpha interferon (IFN-α) and IFN-β are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation.

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