Regulatory functions of 5' terminal sequences in mRNA synthesis, translation and decay

Purpose: The 5' end of a mRNA is a target for key factors in mRNA translation and mRNA destabilization, whereas its sequence is determined by the exact site where transcription initiates. The goal of this study is to see how different 5' end sequences affect mRNA levels, mRNA translation and mRNA stability. Methods: We expressed a reporter Renilla luciferase gene of which the first nucleotides at its 5' end were random 7-mer sequences in HeLa cells. After treatment with RNA polymerase inhibitor Actinomycin D, mTOR inhibitor Torin 1, or vehicle (DMSO) cells were lysed and the reporters 5' ends were sequenced, either directly or after separating strongly translated and poorly translated mRNA molecules on a sucrose gradient. Results: We found that the first 7 nucleotides of a mRNA have a strong impact on mRNA translation and its regulation and on mRNA stability. Overall design: Systematic study of the effect of the 5' end sequence of a mRNA on its expression, translation and stability.