Analysis of translation initiation using a translation control reporter system.
The study of translational control has become increasingly important, as aberrant translation has been linked to the etiology of human diseases. Nevertheless, a convenient research tool to measure and quantify cellular translational activity has not been developed to date. Here we present a translation control reporter system (TCRS) for straightforward and accurate analysis of cellular translational activity. Our method relies on the expression of two unique reporter peptides from a single messenger RNA transcript. Using TCRS-expressing cell lines, changes in initiation of translation have been detected in response to translationally active drugs. Accordingly, TCRS may promote the discovery of novel agents that modulate translation. TCRS may also be used in the identification of signal transduction pathways that impinge on translation control. Furthermore, the modular design allows the exchange of regulatory cassettes for the examination of other putative cis-regulatory mRNA elements. The time required for the procedure depends on whether transient TCRS expression is used or stable TCRS-expressing cell lines have to be produced and will range from 5 to 14 d, respectively.
Wiesenthal
Volker
V
Max Delbrück Centre for Molecular Medicine, Robert Roessle Str. 10, D-13092 Berlin, Germany.
Leutz
Achim
A
Calkhoven
Cornelis F
CF
D002460:Cell Line; D017930:Genes, Reporter / Q000235:genetics*; D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*
D012333:RNA, Messenger