Genetics of the DST-mediated mRNA decay pathway using a transgene-based selection

2004 ◽  
Vol 32 (4) ◽  
pp. 575-577 ◽  
Author(s):  
P. Lidder ◽  
M.A. Johnson ◽  
M.L. Sullivan ◽  
D.M. Thompson ◽  
M.A. Pérez-Amador ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mrna Decay ◽  
Marker Gene ◽  
Mrna Degradation ◽  
Test Case ◽  
Sequence Element ◽  
Technical Aspects ◽  
Screenable Marker ◽  
Post Transcriptional Regulation ◽  
Selection Of

mRNA sequences that control abundance, localization and translation initiation have been identified, yet the factors that recognize these sequences are largely unknown. In this report, a transgene-based strategy designed to isolate mutants of Arabidopsis thaliana that fail to recognize these sequences is described. In this strategy, a selectable gene and a screenable marker gene are put under the control of the sequence element being analysed and mutants are selected with altered abundance of the corresponding marker RNAs. The selection of mutants deficient in recognition of the DST (downstream) mRNA degradation signal is used as a test-case to illustrate some of the technical aspects that have facilitated success. Using this strategy, we report the isolation of a new mutant, dst3, deficient in the DST-mediated mRNA decay pathway. The targeted genetic strategy described circumvents certain technical limitations of biochemical approaches. Hence, it provides a means to investigate a variety of other mechanisms responsible for post-transcriptional regulation.

scholarly journals Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo

PLoS Genetics ◽  
2018 ◽  
Vol 14 (12) ◽  
pp. e1007806 ◽  
Author(s):  
Quira Zeidan ◽  
Feng He ◽  
Fan Zhang ◽  
Hongen Zhang ◽  
Allan Jacobson ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Mrna Decay

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

2005 ◽  
Vol 32 (8) ◽  
pp. 671 ◽  
Author(s):  
Song Chen ◽  
Christopher A. Helliwell ◽  
Li-Min Wu ◽  
Elizabeth S. Dennis ◽  
Narayana M. Upadhyaya ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Marker Gene ◽  
Direct Repeat ◽  
Transgene Silencing ◽  
Selective Marker ◽  
Hairpin Rna ◽  
Transgenic Lines ◽  
Flanking Sequences ◽  
Dna Vector ◽  
Selection Of

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.

mRNA degradation by processive 3′-5′ exoribonucleases in Vitro and the implications for prokaryotic mRNA decay in Vivo

1991 ◽  
Vol 221 (1) ◽  
pp. 81-95 ◽  
Author(s):  
Robert S. McLaren ◽  
Sarah F. Newbury ◽  
Geoffrey S.C. Dance ◽  
Helen C. Causton ◽  
Christopher F. Higgins
Keyword(s):  
Mrna Decay ◽  
Mrna Degradation

scholarly journals Osteogenic differentiation potential and marker gene expression of different porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media

2020 ◽  
Author(s):  
Sangeetha Kannan ◽  
Jyotirmoy Ghosh ◽  
Sujoy K. Dhara
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Marker Gene ◽  
Microscopical Examination ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Basal Media ◽  
Selection Of

AbstractMultipotent porcine mesenchymal stem cells (pMSC) are indispensable for research and therapeutic use. Derivation and culture media might affect the selection of MSC subpopulation and thus the differentiation potential of cells. In this study we evaluated the effects of αMEM, aDMEM, M199, αMEM/M199, aDMEM/M199 and αMEM/aDMEM media on porcine bone marrow MSC derivation; pre-differentiation expression of ALP, COL1A1, SPP1 and BGLAP osteogenic marker genes at passage 5 and 10 pMSC; and differentiation potential of passage 5 pMSC. Morphological changes and matrix formation in osteogenic cells were evaluated by microscopical examination and calcium deposit in osteocytes was confirmed by Alizarin Red S staining. Results indicated media independent selection of different bone marrow MSC subpopulations with different surface marker gene expressions. Many pMSC subpopulations in different media had CD14+ expressing cells. We also observed basal media dependent changes in osteogenic markers expression and differentiation potential of pMSC. The αMEM/aDMEM media grown pMSC showed best osteogenic differentiation potential. We thus recommended the testing of αMEM/aDMEM mixed media in other species for pre-differentiation MSC culture that are intended for better osteogenic differentiation.SummaryPre-differentiation basal media influence osteogenic differentiation potential of mesenchymal stem cells (MSC). Among the tested media, αMEM/aDMEM was the best for pre-differentiation porcine MSC culture intending to use in osteogenesis.

scholarly journals Selective inhibition of the p38α MAPK–MK2 axis inhibits inflammatory cues including inflammasome priming signals

2018 ◽  
Vol 215 (5) ◽  
pp. 1315-1325 ◽  
Author(s):  
Chun Wang ◽  
Susan Hockerman ◽  
E. Jon Jacobsen ◽  
Yael Alippe ◽  
Shaun R. Selness ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Clinical Trials ◽  
Mrna Decay ◽  
Selective Inhibition ◽  
Mrna Degradation ◽  
Cytokine Expression ◽  
Clinical Translation ◽  
Tnf Α ◽  
P38α Mapk ◽  
Nlrp3 Expression

p38α activation of multiple effectors may underlie the failure of global p38α inhibitors in clinical trials. A unique inhibitor (CDD-450) was developed that selectively blocked p38α activation of the proinflammatory kinase MK2 while sparing p38α activation of PRAK and ATF2. Next, the hypothesis that the p38α–MK2 complex mediates inflammasome priming cues was tested. CDD-450 had no effect on NLRP3 expression, but it decreased IL-1β expression by promoting IL-1β mRNA degradation. Thus, IL-1β is regulated not only transcriptionally by NF-κB and posttranslationally by the inflammasomes but also posttranscriptionally by p38α–MK2. CDD-450 also accelerated TNF-α and IL-6 mRNA decay, inhibited inflammation in mice with cryopyrinopathy, and was as efficacious as global p38α inhibitors in attenuating arthritis in rats and cytokine expression by cells from patients with cryopyrinopathy and rheumatoid arthritis. These findings have clinical translation implications as CDD-450 offers the potential to avoid tachyphylaxis associated with global p38α inhibitors that may result from their inhibition of non-MK2 substrates involved in antiinflammatory and housekeeping responses.

scholarly journals c-Myc steers translation in lymphoma

2019 ◽  
Vol 216 (7) ◽  
pp. 1471-1473 ◽  
Author(s):  
Marie Cargnello ◽  
Ivan Topisirovic
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Mrna Translation ◽  
Lymphoma Cells ◽  
Master Regulators ◽  
Selection Of

Members of the MYC family of oncogenes are master regulators of mRNA translation. In this issue of JEM, Singh et al. (https://doi.org/10.1084/jem.20181726) demonstrate that c-Myc governs protein synthesis in lymphoma cells by interfering with SRSF1- and RBM42-mediated suppression of mRNA translation and by altering selection of translation initiation sites.

scholarly journals mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colony-stimulating factor transcripts: different deadenylation kinetics and uncoupling from translation.

1995 ◽  
Vol 15 (10) ◽  
pp. 5777-5788 ◽  
Author(s):  
C Y Chen ◽  
N Xu ◽  
A B Shyu
Keyword(s):  
Mammalian Cells ◽  
Mrna Decay ◽  
Rna Stability ◽  
Mrna Degradation ◽  
Colony Stimulating Factor ◽  
Turnover Rates ◽  
Cis Acting ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Poly(A) tail removal is a critical first step in the decay pathway for many yeast and mammalian mRNAs. Poly(A) shortening rates can be regulated by cis-acting sequences within the transcribed portion of mRNA, which in turn control mRNA turnover rates. The AU-rich element (ARE), found in the 3' untranslated regions of many highly labile mammalian mRNAs, is a well-established example of this type of control. It represents the most widespread RNA stability determinant among those characterized in mammalian cells. Here, we report that two structurally different AREs, the c-fos ARE and the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE, both direct rapid deadenylation as the first step in mRNA degradation, but by different kinetics. For c-fos-ARE-mediated decay, the mRNA population undergoes synchronous poly(A) shortening and is deadenylated at the same rate, implying the action of distributive or nonprocessive ribonucleolytic digestion of poly(A) tails. In contrast, the population of granulocyte-macrophage colony-stimulating factor ARE-containing mRNAs is deadenylated asynchronously, with the formation of fully deadenylated intermediates, consistent with the action of processive ribonucleolytic digestion of poly(A) tails. An important general implication of this finding is that different RNA-destabilizing elements direct deadenylation either by modulating the processivity at which a single RNase functions or by recruiting kinetically distinct RNases. We have also employed targeted inhibition of translation initiation to demonstrate that the RNA-destabilizing function of both AREs can be uncoupled from translation by ribosomes. In addition, a blockade of ongoing transcription has been used to further probe the functional similarities and distinctions of these two AREs. Our data suggest that the two AREs are targets of two distinct mRNA decay pathways. A general model for ARE-mediated mRNA degradation involving a potential role for certain heterogeneous nuclear ribonucleoproteins and ARE-binding proteins is proposed.

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