Multifunctional Roles for the Protein Translocation Machinery in RNA Anchoring to the Endoplasmic Reticulum [RNA]

July 25th, 2014 by Jagannathan, S., Hsu, J. C.- C., Reid, D. W., Chen, Q., Thompson, W. J., Moseley, A. M., Nicchitta, C. V.

mRNAs encoding cytosolic and signal sequence-bearing proteins are translated by free and endoplasmic reticulum (ER)-bound ribosomes, respectively. Recent ribosome footprinting studies have also demonstrated translation of cytosolic protein-encoding mRNAs on ER-bound ribosomes, findings that raise important questions regarding the mechanism of ribosome and mRNA localization and association with the ER. Using a semi- intact HeLa cell model, we performed a polysome solubilization screen and identified conditions that biochemically distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. RNA-protein UV photocrosslinking studies revealed numerous ER integral membrane proteins with RNA binding activity, consistent with direct RNA anchoring functions. Quantitative proteomic analyses of HeLa cytosolic and two classes of ER-bound polysomes identified translocon components as selective polysome-interacting proteins. Notably, the Sec61 complex was highly enriched in polysomes engaged in the translation of endomembrane organelle proteins whereas whereas translocon accessory proteins such as ribophorin I were present in all subpopulations of ER-associated polysomes. Analyses of the protein composition of oligo(dT)-selected UV photocrosslinked protein-RNA adducts identified Sec61α , β and ribophorin I, suggesting roles for the protein translocation and modification machinery in mRNA anchoring to the ER. We propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable a transcriptome-wide function for the ER in cellular protein synthesis.