A translation control reporter system (TCRS) for the analysis of translationally controlled processes in the vertebrate cell.
Regulation of translation is critical for the accurate expression of a broad variety of genes that function in cell cycle progression and cell differentiation, as well as in the adaptation to cellular stress. The aetiologies of a number of human diseases, including cancer, have been linked to mutations in genes that control mRNA translation, or in cis-regulatory mRNA-sequences. Therefore, research on translational control and its therapeutic appliance has become most important. However, to date only a limited number of therapeutic drugs are known to affect translational control. Here we describe a novel, straightforward approach for the detection of cellular translational activity. We developed a Translational Control Reporter System (TCRS), which utilizes the cis-regulatory upstream open reading frame (uORF) from the c/ebpalpha locus to direct the translation of a dual reporter gene into two unique reporter peptides. The peptides contain a pre-pro-trypsin (PPT) signal for secretion into the medium and distinct immunogenic epitopes for detection and quantification purposes. TCRS-peptide expression levels reflect changes of translation initiation induced by serum growth factors, drugs or translation factor mutants. TCRS can be tailored to various research settings and the system may accomplish a broad application to uncover links between translational control and drugs.
Max Delbrück Centre for Molecular Medicine Robert Rössle Strasse 10, D-13092 Berlin, Germany.
Volker
V
Wiesenthal
Achim
A
Leutz
Cornelis F
CF
Calkhoven
D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D016366:Open Reading Frames; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects*; D010455:Peptides / Q000032:analysis / Q000737:chemistry
D039642:Eukaryotic Initiation Factors; D006133:Growth Substances; D010455:Peptides