HeLa cells cultivated in 6-well plates were transfected with plasmid DNA using DreamFect transfection reagent.
This article demonstrates the high efficiency of DreamFect from OZ Biosciences for DNA transfection into cell lines.article reference: PLoS One. 2014 Aug 22;9(8):e104391
Eukaryotic Initiation Factor 4G Suppresses Nonsense-Mediated mRNA Decay by Two Genetically Separable Mechanisms.
Abstract
Nonsense-mediated
mRNA decay (NMD), which is best known for degrading mRNAs with
premature termination codons (PTCs), is thought to be triggered by
aberrant translation termination at stop codons located in an
environment of the mRNP that is devoid of signals necessary for proper
termination. In mammals, the cytoplasmic poly(A)-binding protein 1
(PABPC1) has been reported to promote correct termination and therewith
antagonize NMD by interacting with the eukaryotic
release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which
proteins of interest are recruited as MS2 fusions to a NMD reporter
transcript, we show that the three N-terminal RNA recognition motifs
(RRMs) of PABPC1 are sufficient to antagonize NMD, while the
eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of
PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses
NMD. We identified the interactions of the eIF4G N-terminus with PABPC1
and the eIF4G core domain with eIF3 as two genetically separable
features that independently enable tethered eIF4G to inhibit NMD.
Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in
translation termination and NMD suppression, and they provide
additional evidence for a tight coupling between translation termination
and initiation.
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