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J Biol Chem. 2014 Sep 12;289(37):25907-24. doi: 10.1074/jbc.M114.580688. Epub 2014 Jul 25.

Multifunctional roles for the protein translocation machinery in RNA anchoring to the endoplasmic reticulum.

Author information

1
From the Departments of Cell Biology and.
2
Biochemistry and.
3
the Duke Proteomics Core Facility, Duke University Medical Center, Durham, North Carolina 27710.
4
From the Departments of Cell Biology and Biochemistry and christopher.nicchitta@duke.edu.

Abstract

Signal sequence-encoding mRNAs undergo translation-dependent localization to the endoplasmic reticulum (ER) and at the ER are anchored via translation on Sec61-bound ribosomes. Recent investigations into the composition and membrane association characteristics of ER-associated mRNAs have, however, revealed both ribosome-dependent (indirect) and ribosome-independent (direct) modes of mRNA association with the ER. These findings raise important questions regarding our understanding of how mRNAs are selected, localized, and anchored to the ER. Using semi-intact tissue culture cells, we performed a polysome solubilization screen and identified conditions that distinguish polysomes engaged in the translation of distinct cohorts of mRNAs. To gain insight into the molecular basis of direct mRNA anchoring to the ER, we performed RNA-protein UV photocross-linking studies in rough microsomes and demonstrate that numerous ER integral membrane proteins display RNA binding activity. Quantitative proteomic analyses of HeLa cytosolic and ER-bound polysome fractions 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 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 photocross-linked ER protein-RNA adducts identified Sec61α,β and ribophorin I as ER-poly(A) mRNA-binding proteins, suggesting unexpected roles for the protein translocation and modification machinery in mRNA anchoring to the ER. In summary, we propose that multiple mechanisms of mRNA and ribosome association with ER operate to enable an mRNA transcriptome-wide function for the ER in protein synthesis.

KEYWORDS:

Endoplasmic Reticulum (ER); Protein Translocation; RNA Localization; RNA-Protein Interaction; Ribosome; Signal Recognition Particle (SRP)

PMID:
25063809
PMCID:
PMC4162190
DOI:
10.1074/jbc.M114.580688
[Indexed for MEDLINE]
Free PMC Article

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