Abstract
In eukaryotic cells, the spatial regulation of protein expression is frequently conferred through the coupling of mRNA localization and the local control of translation. mRNA localization to the endoplasmic reticulum (ER) is a prominent example of such regulation and serves a ubiquitous role in segregating the synthesis of secretory and integral membrane proteins to the ER. Recent genomic and biochemical studies have now expanded this view to suggest a more substantial role for the ER cellular protein synthesis. We have utilized cell fractionation and ribosome profiling to obtain a genomic survey of the subcellular organization of mRNA translation and report that ribosomal loading of mRNAs, a proxy for mRNA translation, is biased to the ER. Notably, ER-associated mRNAs encoding both cytosolic and topogenic signal-encoding proteins display similar ribosome loading densities, suggesting that ER-associated ribosomes serve a global role in mRNA translation. We propose that the partitioning of mRNAs and their translation between the cytosol and ER compartments may represent a novel mechanism for the post-transcriptional regulation of gene expression.
Highlights
MRNA translation is compartmentalized between cytosolic and endoplasmic reticulum (ER)-bound ribosomes
These findings indicate that the subcellular organization of mRNA translation may be more complex than generally envisioned and suggest broader roles for the ER in the expression of the mRNA transcriptome. Consistent with this view, ER-associated ribosomes were demonstrated to be competent for de novo initiation [16], to polymerize amino acids with similar kinetics as their cytosolic counterparts [17], and to retain their association with the ER upon termination [18]. These findings demonstrate that the mRNA translation cycle can operate on the ER without the obligatory trafficking of ribosomes and/or mRNAs from the cytosol to the ER and raise a number of key questions regarding how mRNA translation is segregated between the cytosol and ER compartments and whether translation is subject to compartment-specific regulation
To quantify subcellular ribosome quantities and distributions, cell cultures were metabolically labeled to steady state with [3H]uridine, and the mRNAassociated ribosomes were purified by native chromatography on oligo(dT) [7] cellulose resin and quantified by scintillation spectrometry (Fig. 1C)
Summary
Cell Culture and Fractionation—HEK293 cells were grown in DMEM supplemented with 10% FBS. Gels were stained with SYBR Gold (Invitrogen), revealing an ϳ35-nt band that was absent when ribosomes were dissociated with EDTA prior to nuclease digestion (supplemental Fig. S1) This region of the gel was excised, and RNA was extracted by homogenizing the gel in 100 mM NH4HCO3, freezing to Ϫ80 °C, rapidly reheating to 95 °C, and incubating the gel homogenate, with mixing, for 3 h at room temperature. Total RNA samples were treated with RiboMinus (Invitrogen) and prepared for bar-coded SOLiD sequencing according to the manufacturer’s instructions, where total mRNA was treated with RNase III and prepared for sequencing as described above In this run, 35 million 50-base reads were obtained between the two samples. Significance was determined using Benjamini-Hochberg multiple hypothesis correction from a p value cutoff of 0.05
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