In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell.
The process of mRNA localization typically utilizes cis-targeting elements and trans-recognition factors to direct the compartmental organization of translationally suppressed mRNAs. mRNA localization to the endoplasmic reticulum (ER), in contrast, occurs via a co-translational, signal sequence/signal recognition particle (SRP)-dependent mechanism. We have utilized cell fractionation/cDNA microarray analysis, shRNA-mediated suppression of SRP expression, and mRNA reporter construct studies to define the role of the SRP pathway in ER-directed mRNA localization. Cell fractionation studies of mRNA partitioning between the cytosol and ER demonstrated the expected enrichment of cytosolic/nucleoplasmic protein-encoding mRNAs and secretory/integral membrane protein-encoding mRNAs in the cytosol and ER fractions, respectively, and identified a subpopulation of cytosolic/nucleoplasmic protein-encoding mRNAs in the membrane-bound mRNA pool. The latter finding suggests a signal sequence-independent pathway of ER-directed mRNA localization. Extending from these findings, mRNA partitioning was examined in stable SRP54 shRNA knockdown HeLa cell lines. shRNA-directed reductions in SRP did not globally alter mRNA partitioning patterns, although defects in membrane protein processing were observed, further suggesting the existence of multiple pathways for mRNA localization to the ER. ER localization of GRP94-encoding mRNA was observed when translation was disabled by mutation of the start codon/insertion of a 59UTR stem-loop structure or upon deletion of the encoded signal sequence. Combined, these data indicate that the mRNA localization to the ER can be conferred independent of the signal sequence/SRP pathway and suggest that mRNA localization to the ER may utilize cis-encoded targeting information.
This study reveals that mRNAs are partitioned between the cytosol and endoplasmic reticulum (ER) compartments in a hierarchical manner and identifies a prominent role for the ER in global protein synthesis. Two modes of mRNA association with the ER are defined: ribosome dependent and ribosome independent.
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