DNA plasmids were transiently transfected into HeLa cells with DreamFect
This article demonstrate the efficiency of Dreamfect transfection reagent from OZ Biosciences to efficiently deliver plasmid into HeLa cells.article reference: RNA. 2013 Aug 20.
Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways.Metze S, Herzog VA, Ruepp MD, Mühlemann O.
Abstract
Nonsense-mediated
mRNA decay (NMD) is a eukaryotic post-transcriptional gene regulation
mechanism that eliminates mRNAs with the termination codon (TC) located
in an unfavorable environment for efficient translation termination. The
best-studied NMD-targeted mRNAs contain premature termination codons
(PTCs); however, NMD regulates even many physiological mRNAs. An
exon-junction complex (EJC) located downstream from a TC acts as an
NMD-enhancing signal, but is not generally required for NMD. Here, we
compared these "EJC-enhanced" and "EJC-independent" modes of NMD with
regard to their requirement for seven known NMD factors in human cells
using two well-characterized NMD reporter genes (immunoglobulin μ and
β-Globin) with or without an intron downstream from the PTC. We show
that both NMD modes depend on UPF1 and SMG1, but detected
transcript-specific differences with respect to the requirement for UPF2
and UPF3b, consistent with previously reported UPF2- and
UPF3-independent branches of NMD. In addition and contrary to
expectation, a higher sensitivity of EJC-independent NMD to reduced UPF2
and UPF3b concentrations was observed. Our data further revealed a
redundancy of the endo- and exonucleolytic mRNA degradation pathways in
both modes of NMD. Moreover, the relative contributions of both decay
pathways differed between the reporters, with PTC-containing
immunoglobulin μ transcripts being preferentially subjected to
SMG6-mediated endonucleolytic cleavage, whereas β-Globin transcripts
were predominantly degraded by the SMG5/SMG7-dependent pathway. Overall,
the surprising heterogeneity observed with only two NMD reporter pairs
suggests the existence of several mechanistically distinct branches of
NMD in human cells.
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