Summary Tail-anchored (TA) proteins are characterized by a C-terminal transmembrane region that mediates posttranslational insertion into the membrane of the endoplasmic reticulum. We have investigated the requirements for membrane insertion of three TA proteins, RAMP4, Sec61β and cytochrome b5. We show here that newly synthesized RAMP4 and Sec61β can accumulate in a cytosolic, soluble complex with the ATPase Asna-1/TRC40 before insertion into ER-derived membranes. Membrane insertion of these TA proteins is stimulated by ATP, sensitive to redox conditions and blocked by alkylation of SH groups by N-ethylmaleimide (NEM). In contrast, membrane insertion of cytochrome b5 is not found to be mediated by Asna-1, not stimulated by ATP and not affected by NEM or an oxidative environment. Asna-1 mediated pathway of membrane insertion of RAMP4 and Sec61β may relate to functions of these proteins in the ER stress response.

In mammalian cells, AU-rich elements (AREs) are well known regulatory sequences located in the 3′ untranslated region (UTR) of many short-lived mRNAs. AREs cause mRNAs to be degraded rapidly and thereby suppress gene expression at the posttranscriptional level. Based on the number of AUUUA pentamers, their proximity, and surrounding AU-rich regions, we generated an algorithm termed AREScore that identifies AREs and provides a numerical assessment of their strength. By analyzing the AREScore distribution in the transcriptomes of 14 metazoan species, we provide evidence that AREs were selected for in several vertebrates and Drosophila melanogaster. We then measured mRNA expression levels genome-wide to address the importance of AREs in SL2 cells derived from D. melanogaster hemocytes. Tis11, a zinc finger RNA–binding protein homologous to mammalian tristetraprolin, was found to target ARE–containing reporter mRNAs for rapid degradation in SL2 cells. Drosophila mRNAs whose expression is elevated upon knock down of Tis11 were found to have higher AREScores. Moreover high AREScores correlate with reduced mRNA expression levels on a genome-wide scale. The precise measurement of degradation rates for 26 Drosophila mRNAs revealed that the AREScore is a very good predictor of short-lived mRNAs. Taken together, this study introduces AREScore as a simple tool to identify ARE–containing mRNAs and provides compelling evidence that AREs are widespread regulatory elements in Drosophila.