Mechanisms ofmRNA partitioning in mammalian cells: The role of protein synthesis in mRNA localization at the endoplasmic reticulum.

摘要

In eukaryotic cells, protein synthesis is compartmentalized: mRNAs encoding signal sequences are translated on endoplasmic reticulum (ER)-bound ribosomes, while mRNAs lacking encoded signal sequences are translated on cytosolic ribosomes. Only those ribosomes that have translated a signal sequence peptide are selected for trafficking to and continued translation at the ER. Membrane-bound ribosome release is thought to be coincident with protein synthesis termination, creating a model in which ribosome cycling to and from the ER is protein-synthesis dependent. The predictions of this model---(1) all ER-localized mRNAs encode a signal sequence, and (2) ribosome-mRNA complexes are released from the ER upon termination---were tested, and the results of these studies are presented in this report.; To test the first prediction, the composition of the ER-associated mRNA population was analyzed. Tissue culture cells were biochemically fractionated to yield purified ER and assessed for the presence of individual messages. These studies demonstrated that mRNAs encoding soluble proteins are ER-associated and translated on membrane-bound polysomes. In situ hybridization experiments on intact cells revealed mRNAs encoding soluble proteins on the ER, confirming results attained from biochemical analyses. These results suggest that cells possess a novel pathway to localize mRNAs to the ER, independent of encoded signal sequence.; The role of protein synthesis in ER-localization of mRNAs was addressed using pharmacological and physiological approaches. ER fractions purified from cells treated with pharmacological inhibitors were analyzed to determine whether individual messages were retained at or released from the ER upon termination. These studies revealed the retention of mRNAs on the ER following termination of protein synthesis. As an alternative approach, cells were infected with coxsackie B3 virus (CBV), which inhibits cap-dependent translation, blocking the majority of cellular protein synthesis. In cells infected with CBV, mRNAs were released from cytosolic ribosomes while ER-bound ribosomes maintained their association with mRNAs. These data suggest a role for the ER as a privileged site of protein synthesis during cell stress when general protein synthesis is compromised. Characterization of these previously unknown ER functions described here will contribute towards understanding the role of the ER under both normal and disease states.