DEGRADATION OF MESSENGER RNA IN MAMMALIAN CELLS

1972 ◽  
Vol 71 (2_Suppla) ◽  
pp. S369-S380 ◽  
Author(s):  
Francis T. Kenney ◽  
Kai-Lin Lee ◽  
Charles D. Stiles
Keyword(s):  
Messenger Rna ◽  
Mammalian Cells ◽  
Messenger Rnas ◽  
Tyrosine Transaminase

ABSTRACT Analyses of the response of hydrocortisone-induced tyrosine transaminase in cultured H-35 cells to inhibitors of translation (cycloheximide, puromycin) suggest: (1) that bound ribosomes stabilize messenger RNA in vivo; (2) that messenger is degraded at a rate determined by the rate of translation. Since specific messenger RNAs of mammalian cells are degraded at quite different rates, there may be extensive heterogeneity either in the rate at which ribosomes traverse different messengers or in the number of ribosomes which translate specific messenger RNAs.

scholarly journals SMG-1 Is a Phosphatidylinositol Kinase-Related Protein Kinase Required for Nonsense-Mediated mRNA Decay in Caenorhabditis elegans

2004 ◽  
Vol 24 (17) ◽  
pp. 7483-7490 ◽  
Author(s):  
Andrew Grimson ◽  
Sean O'Connor ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Null Alleles ◽  
Dependent Manner ◽  
Messenger Rnas ◽  
Phosphatidylinositol Kinase ◽  
Nonsense Mediated Mrna Decay

ABSTRACT Eukaryotic messenger RNAs containing premature stop codons are selectively and rapidly degraded, a phenomenon termed nonsense-mediated mRNA decay (NMD). Previous studies with both Caenohabditis elegans and mammalian cells indicate that SMG-2/human UPF1, a central regulator of NMD, is phosphorylated in an SMG-1-dependent manner. We report here that smg-1, which is required for NMD in C. elegans, encodes a protein kinase of the phosphatidylinositol kinase superfamily of protein kinases. We identify null alleles of smg-1 and demonstrate that SMG-1 kinase activity is required in vivo for NMD and in vitro for SMG-2 phosphorylation. SMG-1 and SMG-2 coimmunoprecipitate from crude extracts, and this interaction is maintained in smg-3 and smg-4 mutants, both of which are required for SMG-2 phosphorylation in vivo and in vitro. SMG-2 is located diffusely through the cytoplasm, and its location is unaltered in mutants that disrupt the cycle of SMG-2 phosphorylation. We discuss the role of SMG-2 phosphorylation in NMD.

scholarly journals Specific combinations of SR proteins associate with single pre-messenger RNAs in vivo and contribute different functions

2009 ◽  
Vol 184 (4) ◽  
pp. 555-568 ◽  
Author(s):  
Petra Björk ◽  
ShaoBo Jin ◽  
Jian Zhao ◽  
Om Prakash Singh ◽  
Jan-Olov Persson ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Sr Proteins ◽  
Chironomus Tentans ◽  
Messenger Rnas ◽  
Balbiani Ring ◽  
Functional Differences ◽  
Nucleocytoplasmic Export ◽  
Nascent Transcripts ◽  
Alternative Splicing Factor

Serine/arginine-rich (SR) proteins are required for messenger RNA (mRNA) processing, export, surveillance, and translation. We show that in Chironomus tentans, nascent transcripts associate with multiple types of SR proteins in specific combinations. Alternative splicing factor (ASF)/SF2, SC35, 9G8, and hrp45/SRp55 are all present in Balbiani ring (BR) pre-messenger ribonucleoproteins (mRNPs) preferentially when introns appear in the pre-mRNA and when cotranscriptional splicing takes place. However, hrp45/SRp55 is distributed differently in the pre-mRNPs along the gene compared with ASF/SF2, SC35, and 9G8, suggesting functional differences. All four SR proteins are associated with the BR mRNPs during export to the cytoplasm. Interference with SC35 indicates that SC35 is important for the coordination of splicing, transcription, and 3′ end processing and also for nucleocytoplasmic export. ASF/SF2 is associated with polyribosomes, whereas SC35, 9G8, and hrp45/SRp55 cosediment with monoribosomes. Thus, individual endogenous pre-mRNPs/mRNPs bind multiple types of SR proteins during transcription, and these SR proteins accompany the mRNA and play different roles during the gene expression pathway in vivo.

scholarly journals Intranuclear binding in space and time of exon junction complex and NXF1 to premRNPs/mRNPs in vivo

2015 ◽  
Vol 211 (1) ◽  
pp. 63-75 ◽  
Author(s):  
Petra Björk ◽  
Jan-Olov Persson ◽  
Lars Wieslander
Keyword(s):  
Messenger Rna ◽  
Protein Complexes ◽  
Spatial Association ◽  
Exon Junction Complex ◽  
Messenger Rnas ◽  
Nonsense Mediated Decay ◽  
Exon Junction ◽  
Eukaryotic Gene ◽  
Core Components

Eukaryotic gene expression requires the ordered association of numerous factors with precursor messenger RNAs (premRNAs)/messenger RNAs (mRNAs) to achieve efficiency and regulation. Here, we use the Balbiani ring (BR) genes to demonstrate the temporal and spatial association of the exon junction complex (EJC) core with gene-specific endogenous premRNAs and mRNAs. The EJC core components bind cotranscriptionally to BR premRNAs during or very rapidly after splicing. The EJC core does not recruit the nonsense-mediated decay mediaters UPF2 and UPF3 until the BR messenger RNA protein complexes (mRNPs) enter the interchromatin. Even though several known adapters for the export factor NXF1 become part of BR mRNPs already at the gene, NXF1 binds to BR mRNPs only in the interchromatin. In steady state, a subset of the BR mRNPs in the interchromatin binds NXF1, UPF2, and UPF3. This binding appears to occur stochastically, and the efficiency approximately equals synthesis and export of the BR mRNPs. Our data provide unique in vivo information on how export competent eukaryotic mRNPs are formed.

scholarly journals In vitro translation of the intermediate filament proteins desmin and vimentin

1981 ◽  
Vol 1 (4) ◽  
pp. 303-309
Author(s):  
C M O'Connor ◽  
D J Asai ◽  
C N Flytzanis ◽  
E Lazarides
Keyword(s):  
Smooth Muscle ◽  
Intermediate Filament ◽  
Messenger Rna ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
In Vitro Translation ◽  
Messenger Rnas ◽  
Intermediate Filament Proteins

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.

scholarly journals Regulated Ire1-dependent decay of messenger RNAs in mammalian cells

2009 ◽  
Vol 186 (3) ◽  
pp. 323-331 ◽  
Author(s):  
Julie Hollien ◽  
Jonathan H. Lin ◽  
Han Li ◽  
Nicole Stevens ◽  
Peter Walter ◽  
...  
Keyword(s):  
Er Stress ◽  
Messenger Rna ◽  
Protein Misfolding ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Nuclease Activity ◽  
Messenger Rnas ◽  
Wild Type ◽  
Nuclease Domain ◽  
Active Transcription

Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box–binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.

scholarly journals Expression of microRNA during bovine adipogenesis

2010 ◽  
Vol 1 (1) ◽  
pp. 12 ◽  
Author(s):  
Scott L. Pratt ◽  
T. Ashley Burns ◽  
Erin Curry ◽  
Susan K. Duckett
Keyword(s):  
Messenger Rna ◽  
Differentially Expressed ◽  
Transcriptional Level ◽  
Messenger Rnas ◽  
Phosphatase And Tensin Homolog ◽  
Tensin Homolog ◽  
Expression Of Genes ◽  
Bovine Adipocytes

Studies have recently indicated that the adipogenic process and the expression of genes involved in lipid metabolism may be regulated in part at the post-transcriptional level by a class of small RNA called microRNA (miRNA). The objectives of this study were to i) determine if miRNAs are differentially expressed, and ii) evaluate expression of known miRNA targets in bovine adipocytes. Bovine adipose samples were collected from castrated males fattened on a high concentrate diet (C) or pasture (PA) and were frozen in liquid nitrogen and stored at -80°C, or used to generate primary stromal-vascular cells (SV). SV cells were cultured to confluence (Control) or differentiated at confluence and harvested 2 (D2), 6 (D6), or 12 (D12) days post-confluence. A 3x3 microarray analysis was performed comparing Control and differentiated samples. miR-21, -221, and -222 (P less than 0.05) were differentially expressed. qRT-PCR was conducted using the<em> in vitro</em> samples, and all three miRNAs were down regulated on D2 (P less than 0.05). miR-221 and -222 were decreased on D6 compared to Control (P less than 0.05), but only miR-222 expression was decreased at D12 (P less than 0.05) compared to Control. miR-21 increased in expression compared to Control on D12 (P less than 0.05). <em>In vivo</em>, only miR-21 expression was affected and it was reduced in PA compared to C fat samples (P less than 0.05). Two targets of miR-21 are Programmed Cell Death Protein 4 (PDCD4) and Phosphatase and Tensin Homolog (PTEN), and neither messenger RNA was differentially expressed<em> in vitro</em> (P greater than 0.05), but both messenger RNAs were elevated for PA compared to C (P less than 0.05). These data show that miRNAs are differentially expressed in adipose cells and tissue, and that miR-21 may be involved in adipocyte function by regulating the translation of PDCD4 and PTEN.

scholarly journals In vitro translation of the intermediate filament proteins desmin and vimentin.

1981 ◽  
Vol 1 (4) ◽  
pp. 303-309 ◽  
Author(s):  
C M O'Connor ◽  
D J Asai ◽  
C N Flytzanis ◽  
E Lazarides
Keyword(s):  
Smooth Muscle ◽  
Intermediate Filament ◽  
Messenger Rna ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
In Vitro Translation ◽  
Messenger Rnas ◽  
Intermediate Filament Proteins

Polyadenylated ribonucleic acid (RNA) was isolated from chicken skeletal and smooth muscle and translated in a cell-free rabbit reticulocyte system. Both types of muscle tissue contain messenger RNAs that code for the intermediate filament proteins desmin and vimentin, and the relative concentrations of the two translation products reflect the prevalence of the two proteins in vivo. Desmin synthesis represents a greater proportion of the total protein synthesis from smooth muscle RNA than from skeletal muscle RNA, whereas the converse is true of vimentin synthesis. Fractionation of the RNA on formamide-containing sucrose gradients before translation indicates that the desmin messenger RNA is larger than the vimentin messenger RNA and contains an extensive noncoding segment. The desmin and vimentin messages code predominantly for the non-phosphorylated forms of desmin and vimentin. However, the ratio of phosphorylated to unphosphorylated forms of the proteins could be increased by adding cyclic adenosine monophosphate-dependent kinase activity to the translation mixtures. These results suggest that desmin and vimentin are each synthesized from a single messenger RNA species and that posttranslational phosphorylation generates the additional isoelectric variants of each which are observed in vivo.

scholarly journals Systemic delivery of factor IX messenger RNA for protein replacement therapy

2017 ◽  
Vol 114 (10) ◽  
pp. E1941-E1950 ◽  
Author(s):  
Suvasini Ramaswamy ◽  
Nina Tonnu ◽  
Kiyoshi Tachikawa ◽  
Pattraranee Limphong ◽  
Jerel B. Vega ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Repeated Administration ◽  
Factor Ix ◽  
Great Promise ◽  
Messenger Rnas ◽  
Systemic Delivery ◽  
Current Standard ◽  
Hepatic Diseases ◽  
Human Factor Ix

Safe and efficient delivery of messenger RNAs for protein replacement therapies offers great promise but remains challenging. In this report, we demonstrate systemic, in vivo, nonviral mRNA delivery through lipid nanoparticles (LNPs) to treat a Factor IX (FIX)-deficient mouse model of hemophilia B. Delivery of human FIX (hFIX) mRNA encapsulated in our LUNAR LNPs results in a rapid pulse of FIX protein (within 4–6 h) that remains stable for up to 4–6 d and is therapeutically effective, like the recombinant human factor IX protein (rhFIX) that is the current standard of care. Extensive cytokine and liver enzyme profiling showed that repeated administration of the mRNA–LUNAR complex does not cause any adverse innate or adaptive immune responses in immune-competent, hemophilic mice. The levels of hFIX protein that were produced also remained consistent during repeated administrations. These results suggest that delivery of long mRNAs is a viable therapeutic alternative for many clotting disorders and for other hepatic diseases where recombinant proteins may be unaffordable or unsuitable.

scholarly journals Addiction of B Cell Lymphomas to microRNA-17-92 Is Mediated By a Single Target Gene

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1258-1258
Author(s):  
Katia Schoeler ◽  
Teresa Mittermeier ◽  
Andreas Villunger ◽  
Klaus Rajewsky ◽  
Verena Labi
Keyword(s):  
B Cells ◽  
B Cell ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
B Cell Lymphoma ◽  
Messenger Rnas ◽  
B Cell Lymphomas ◽  
Apoptosis Pathway

Mature microRNAs (miRNA) are short non-coding RNAs that regulate gene expression by binding to messenger RNAs (mRNA) in a sequence-specific manner, causing translational repression and/or mRNA decay. The mammalian genome harbors thousands of miRNA genes, many of which are organized into transcriptionally co-regulated clusters such as miR-17-92. Knockout of the miR-17-92 cluster gene in mice blocked B lymphopoiesis, and ectopic miR-17-92 expression sufficed to initiate B cell lymphomas and autoimmunity. In humans, the miR-17-92 gene is commonly amplified or overexpressed via MYC-driven transcription in diffuse large B cell and Burkitt's lymphomas. A computationally predicted shared target of the miR-17-92 miRNAs is the pro-apoptotic BCL-2 family protein BIM, central to life-death decisions in mammalian cells. To clarify the contribution of miR-17-92:Bim interactions to the miR-17-92 knockout and overexpression phenotypes, we engineered a unique in vivo system of conditional mutagenesis of the nine Bim 3'UTR miR-17-92 binding sites. Instead of causing the predicted B cell developmental block, interruption of miR-17-92:Bim interactions produced a selective inability of B cells to resist cellular stress; and prevented lymphocyte hyperplasia caused by Bim haploinsufficiency. Surprisingly, partial genetic disruption of miR-17-92:Bim interactions was sufficient to fully prevent B cell lymphoma formation in two out of three mice using two independent pre-clinical MYC-driven cancer models. This protective effect could be attributed to an increased activity of the mitochondrial apoptosis pathway in pre-malignant B cells, as apoptosis was abolished by concomitant overexpression of an anti-apoptotic BCL-2 protein. MYC-driven B lymphoma cells are addicted to miR-17-92 function. Our data build on these results and strongly suggest that miR-17-92:Bim interactions are vital in this context as acute ablation of miR-17-92:Bim interactions effectively promoted lymphoma cell apoptosis, both in vitro and in vivo. In conclusion, among hundreds of putative miR-17-92 target mRNAs a single direct binding partner is vital for lymphoma development and maintenance, a discovery whose therapeutic exploitation is of major relevance. Disclosures No relevant conflicts of interest to declare.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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