32 research outputs found
Upstream ORF affects MYCN translation depending on exon 1b alternative splicing
<p>Abstract</p> <p>Background</p> <p>The <it>MYCN </it>gene is transcribed into two major mRNAs: one full-length (<it>MYCN) </it>and one exon 1b-spliced (<it>MYCN</it><sup>Î1<it>b</it></sup>) mRNA. But nothing is known about their respective ability to translate the MYCN protein.</p> <p>Methods</p> <p>Plasmids were prepared to enable translation from the upstream (uORF) and major ORF of the two <it>MYCN </it>transcripts. Translation was studied after transfection in neuroblastoma SH-EP cell line. Impact of the upstream AUG on translation was evaluated after directed mutagenesis. Functional study with the two <it>MYCN </it>mRNAs was conducted by a cell viability assay. Existence of a new protein encoded by the <it>MYCN</it><sup>Î1<it>b </it></sup>uORF was explored by designing a rabbit polyclonal antibody against a specific epitope of this protein.</p> <p>Results</p> <p>Both are translated, but higher levels of protein were seen with <it>MYCN</it><sup>Î1<it>b </it></sup>mRNA. An upstream ORF was shown to have positive cis-regulatory activity on translation from <it>MYCN </it>but not from <it>MYCN</it><sup>Î1<it>b </it></sup>mRNA. In transfected SH-EP neuroblastoma cells, high MYCN dosage obtained with <it>MYCN</it><sup>Î1<it>b </it></sup>mRNA translation induces an antiapoptotic effect after serum deprivation that was not observed with low MYCN expression obtained with <it>MYCN </it>mRNA. Here, we showed that MYCNOT: <it>MYCN </it>Overlap Transcript, a new protein of unknown function is translated from the upstream AUG of <it>MYCN</it><sup>Î1<it>b </it></sup>mRNA.</p> <p>Conclusions</p> <p>Existence of upstream ORF in <it>MYCN </it>transcripts leads to a new level of MYCN regulation. The resulting MYCN dosage has a weak but significant anti-apoptotic activity after intrinsic apoptosis induction.</p
Neufassung der DIN 4109 auf der Basis EuropÀischer Regelwerke des Baulichen Schallschutzes
Novel Tetrahydropyran-Based Bacterial Topoisomerase Inhibitors with Potent Anti-Gram Positive Activity and Improved Safety Profile
SingleâMolecule FRET of Membrane Transport Proteins
Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membraneâembedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static highâresolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of stateâofâtheâart smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design