scholarly journals Characterization of IRE1 ribonuclease-mediated mRNA decay in plants using transient expression analyses in rice protoplasts

2016 ◽  
Vol 210 (4) ◽  
pp. 1259-1268 ◽  
Author(s):  
Shimpei Hayashi ◽  
Yuhya Wakasa ◽  
Kenjirou Ozawa ◽  
Fumio Takaiwa
Keyword(s):  
Transient Expression ◽  
Mrna Decay

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

scholarly journals Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay.

1995 ◽  
Vol 15 (4) ◽  
pp. 2231-2244 ◽  
Author(s):  
S Zhang ◽  
M J Ruiz-Echevarria ◽  
Y Quan ◽  
S W Peltz
Keyword(s):  
Mrna Decay ◽  
Sequence Motif ◽  
Nonsense Mutations ◽  
Stem Loop ◽  
Cis Acting ◽  
Stem Loop Structure ◽  
Nonsense Codon ◽  
Nonsense Mediated Mrna Decay

In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.

scholarly journals Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

1996 ◽  
Vol 7 (10) ◽  
pp. 1485-1498 ◽  
Author(s):  
M Ramachandra ◽  
S V Ambudkar ◽  
M M Gottesman ◽  
I Pastan ◽  
C A Hrycyna
Keyword(s):  
Drug Resistance ◽  
Atpase Activity ◽  
Cyclosporin A ◽  
Transient Expression ◽  
Expression System ◽  
Rhodamine 123 ◽  
Wild Type ◽  
P Glycoprotein ◽  
Transient Expression System

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.

Discovery and Characterization of a Eukaryotic Initiation Factor 4A-3-Selective Inhibitor That Suppresses Nonsense-Mediated mRNA Decay

2017 ◽  
Vol 12 (7) ◽  
pp. 1760-1768 ◽  
Author(s):  
Misa Iwatani-Yoshihara ◽  
Masahiro Ito ◽  
Yoshihiro Ishibashi ◽  
Hideyuki Oki ◽  
Toshio Tanaka ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Selective Inhibitor ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Nonsense Mediated Mrna Decay

Characterization of SERCA2b Ca2+–Mg2+ ATPase mRNA decay by nuclear proteins

Cell Calcium ◽  
2007 ◽  
Vol 41 (6) ◽  
pp. 581-592 ◽  
Author(s):  
Tao Chen ◽  
Paromita Ghosh ◽  
Christine M. Misquitta ◽  
Archana Govindan ◽  
Ashok K. Grover
Keyword(s):  
Mrna Decay ◽  
Nuclear Proteins

scholarly journals Detection and Degradation of Nonsense-mediated mRNA Decay Substrates Involve Two Distinct Upf1-bound Complexes

2018 ◽  
Author(s):  
Marine Dehecq ◽  
Laurence Decourty ◽  
Abdelkader Namane ◽  
Caroline Proux ◽  
Joanne Kanaan ◽  
...  
Keyword(s):  
Large Scale ◽  
Mrna Decay ◽  
Affinity Purification ◽  
Rna Degradation ◽  
Degradation Pathway ◽  
Telomere Maintenance ◽  
Mrna Decapping ◽  
Nonsense Mediated Mrna Decay ◽  
Decapping Enzyme

AbstractNonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that in all eukaryotes NMD operates through successive Upf1-bound Detector and Effector complexes.

scholarly journals Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex

2010 ◽  
Vol 39 (1) ◽  
pp. 347-358 ◽  
Author(s):  
Israel S. Fernández ◽  
Akio Yamashita ◽  
Ernesto Arias-Palomo ◽  
Yumi Bamba ◽  
Ruben A. Bartolomé ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Essential Component ◽  
Nonsense Mediated Mrna Decay
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