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Primary Role for Endoplasmic Reticulum-bound Ribosomes in Cellular Translation Identified by Ribosome Profiling*

  • David W. Reid
    Affiliations
    Departments of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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  • Christopher V. Nicchitta
    Correspondence
    To whom correspondence should be addressed. Tel.: 919-684-8948; Fax: 919-684-5481
    Affiliations
    Departments of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

    Departments of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants GM077382 and GM077382-OS31 (to C. V. N.). This work was also supported by a Core Facilities voucher from the Duke University School of Medicine.
    This article contains supplemental Figs. S1–S4 and Tables S1–S3.
Open AccessPublished:December 23, 2011DOI:https://doi.org/10.1074/jbc.M111.312280
      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.

      Introduction

      The translation of mRNAs into proteins occurs primarily on two populations of ribosomes: those free in the cytosol and those bound to the endoplasmic reticulum (ER)
      The abbreviations used are:
      ER
      endoplasmic reticulum
      GO
      gene ontology
      nt
      nucleotide(s).
      (
      • Siekevitz P.
      • Palade G.E.
      A cytochemical study on the pancreas of the guinea pig. I. Isolation and enzymatic activities of cell fractions.
      ,
      • Redman C.M.
      Biosynthesis of serum proteins and ferritin by free and attached ribosomes of rat liver.
      ,
      • Hicks S.J.
      • Drysdale J.W.
      • Munro H.N.
      Preferential synthesis of ferritin and albumin by different populations of liver polysomes.
      ). The landmark studies of Palade and colleagues (
      • Caro L.G.
      • Palade G.E.
      Protein synthesis, storage, and discharge in the pancreatic exocrine cell: an autoradiographic study.
      ,
      • Jamieson J.D.
      • Palade G.E.
      Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex.
      ) demonstrated that membrane-bound ribosomes function in the translation of proteins destined to enter the secretory pathway. Consequently, subsequent investigations into the contributions of ER-bound ribosome to cellular protein synthesis have focused on integral membrane and secretory protein biogenesis and the mechanisms mediating the recruitment of secretory and membrane protein-encoding mRNAs to the ER (
      • Walter P.
      • Blobel G.
      Purification of a membrane-associated protein complex required for protein translocation across the endoplasmic reticulum.
      ,
      • Walter P.
      • Blobel G.
      Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in vitro-assembled polysomes synthesizing secretory protein.
      ,
      • Blobel G.
      • Walter P.
      • Chang C.N.
      • Goldman B.M.
      • Erickson A.H.
      • Lingappa V.R.
      Translocation of proteins across membranes: the signal hypothesis and beyond.
      ).
      Genome-scale studies of mRNA partitioning between the cytosol and ER compartments have demonstrated, somewhat unexpectedly, that the mRNA transcriptome is broadly represented on the ER, with mRNAs that encode secretory and membrane proteins being highly ER-enriched and mRNAs encoding cytosolic/nucleoplasmic proteins displaying overlapping subcellular distributions with a prominent cytosolic enrichment (
      • Chen Q.
      • Jagannathan S.
      • Reid D.W.
      • Zheng T.
      • Nicchitta C.V.
      Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells.
      ,
      • Diehn M.
      • Eisen M.B.
      • Botstein D.
      • Brown P.O.
      Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.
      ,
      • Diehn M.
      • Bhattacharya R.
      • Botstein D.
      • Brown P.O.
      Genome-scale identification of membrane-associated human mRNAs.
      ). Furthermore, in a limited number of cases, mRNAs that lack an encoded topogenic signal were reported to be highly partitioned to the ER (
      • Aragón T.
      • van Anken E.
      • Pincus D.
      • Serafimova I.M.
      • Korennykh A.V.
      • Rubio C.A.
      • Walter P.
      Messenger RNA targeting to endoplasmic reticulum stress signaling sites.
      ,
      • Liao G.
      • Ma X.
      • Liu G.
      An RNA-zipcode-independent mechanism that localizes Dia1 mRNA to the perinuclear ER through interactions between Dia1 nascent peptide and Rho-GTP.
      ,
      • Lerner R.S.
      • Seiser R.M.
      • Zheng T.
      • Lager P.J.
      • Reedy M.C.
      • Keene J.D.
      • Nicchitta C.V.
      Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes.
      ,
      • Stephens S.B.
      • Dodd R.D.
      • Brewer J.W.
      • Lager P.J.
      • Keene J.D.
      • Nicchitta C.V.
      Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation.
      ). Combined, these findings indicate that the subcellular organization of mRNA translation may be more complex then 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 (
      • Potter M.D.
      • Nicchitta C.V.
      Regulation of ribosome detachment from the mammalian endoplasmic reticulum membrane.
      ), to polymerize amino acids with similar kinetics as their cytosolic counterparts (
      • Stephens S.B.
      • Nicchitta C.V.
      Divergent regulation of protein synthesis in the cytosol and endoplasmic reticulum compartments of mammalian cells.
      ), and to retain their association with the ER upon termination (
      • Seiser R.M.
      • Nicchitta C.V.
      The fate of membrane-bound ribosomes following the termination of protein synthesis.
      ). 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. These questions are of substantial importance; several recent studies have shown that ER-associated ribosomes maintain translational activity under stress conditions that elicit a translational inhibition in the cytosol, and so the subcellular partitioning of mRNAs may have a substantial impact on the expression of the encoded proteins (
      • Stephens S.B.
      • Dodd R.D.
      • Brewer J.W.
      • Lager P.J.
      • Keene J.D.
      • Nicchitta C.V.
      Stable ribosome binding to the endoplasmic reticulum enables compartment-specific regulation of mRNA translation.
      ,
      • Unsworth H.
      • Raguz S.
      • Edwards H.J.
      • Higgins C.F.
      • Yagüe E.
      mRNA escape from stress granule sequestration is dictated by localization to the endoplasmic reticulum.
      ,
      • Lerner R.S.
      • Nicchitta C.V.
      mRNA translation is compartmentalized to the endoplasmic reticulum following physiological inhibition of cap-dependent translation.
      ).
      The study of in vivo mRNA translation has been substantially advanced by the recent development of a ribosomal footprinting protocol where ribosome-protected regions of mRNAs are isolated and analyzed by deep sequencing to provide a genome-scale, subcodon resolution survey of ribosomal loading of the transcriptome (
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ,
      • Ingolia N.T.
      • Lareau L.F.
      • Weissman J.S.
      Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.
      ,
      • Guo H.
      • Ingolia N.T.
      • Weissman J.S.
      • Bartel D.P.
      Mammalian microRNAs predominantly act to decrease target mRNA levels.
      ). This method provides a robust proxy for translational activity, is well correlated with protein abundance (
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ), and has been applied in yeast and mammalian systems to reveal important information about the basic properties of translation in each (
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ,
      • Ingolia N.T.
      • Lareau L.F.
      • Weissman J.S.
      Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.
      ,
      • Guo H.
      • Ingolia N.T.
      • Weissman J.S.
      • Bartel D.P.
      Mammalian microRNAs predominantly act to decrease target mRNA levels.
      ). Here, we utilize genome-scale ribosome footprinting of cytosolic and ER-associated polyribosomes to identify the contribution of each compartment to global protein synthesis.

      RESULTS

      To study mRNA distribution and translation on cytosolic and ER-bound polyribosomes, HEK293 cells were fractionated using a previously described sequential detergent extraction protocol that generates highly enriched cytosolic and ER-derived fractions (supplemental Fig. S3) (
      • Lerner R.S.
      • Seiser R.M.
      • Zheng T.
      • Lager P.J.
      • Reedy M.C.
      • Keene J.D.
      • Nicchitta C.V.
      Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes.
      ,
      • Jagannathan S.
      • Nwosu C.
      • Nicchitta C.V.
      Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation.
      ,
      • Stephens S.B.
      • Dodd R.D.
      • Lerner R.S.
      • Pyhtila B.M.
      • Nicchitta C.V.
      Analysis of mRNA partitioning between the cytosol and endoplasmic reticulum compartments of mammalian cells.
      ). To assess polyribosome structure, sucrose density gradient velocity sedimentation studies were performed for cytosol- and ER-derived fractions (Fig. 1, A and B). The polyribosome profiles for both fractions were similar, with abundant polyribosomes present in each. The cytosol fraction contained somewhat higher levels of 40 S and 60 S ribosomal subunits and 80 S ribosomes relative to the polysomal population. To quantify subcellular ribosome quantities and distributions, cell cultures were metabolically labeled to steady state with [3H]uridine, and the mRNA-associated ribosomes were purified by native chromatography on oligo(dT) (
      • Walter P.
      • Blobel G.
      Translocation of proteins across the endoplasmic reticulum. II. Signal recognition protein (SRP) mediates the selective binding to microsomal membranes of in vitro-assembled polysomes synthesizing secretory protein.
      ) cellulose resin and then quantified by scintillation spectrometry (Fig. 1C). Consistent with the quantification of the 80 S monosome/polyribosome profiles illustrated in Fig. 1, A and B, similar quantities of mRNA-associated ribosomes were recovered from each fraction. To quantify total mRNA levels in the cytosol- and ER-derived fractions, total mRNA was selected from each fraction using subtractive hybridization against rRNAs, and quantities were determined by Bioanalyzer analysis. In contrast to ribosomal distributions, total mRNA levels differed in the two fractions, with total mRNA levels being ∼2-fold higher in the cytosol (Fig. 1C). These data suggest that, on average, ribosomes are more densely loaded on ER-bound versus cytosolic mRNAs.
      To evaluate the global translational status of ribosomes in the ER and cytosol, cells were labeled with [35S]Met/Cys for 2 min and then treated with puromycin, which elicits the premature termination of translation and partial polyribosome breakdown (
      • Rabinovitz M.
      • Fisher J.M.
      A dissociative effect of puromycin on the pathway of protein synthesis by Ehrlich ascites tumor cells.
      ,
      • Blobel G.
      • Sabatini D.
      Dissociation of mammalian polyribosomes into subunits by puromycin.
      ). Puromycin treatment elicited breakdown of polyribosomes in both compartments, indicating that both populations of polyribosomes were translationally active (Fig. 2). Corroborating this conclusion, [35S]Met/Cys was incorporated into nascent polypeptides in each polyribosome population (Fig. 2, A and B), and polysomal [35S]Met/Cys was abolished upon addition of puromycin (Fig. 2, C and D). To confirm that mRNAs encoding cytosolic proteins were among the population of actively translating polysomes that were disassembled by puromycin on the ER, semiquantitative PCR targeting ACTB and GAPDH mRNAs, both of which lack topogenic signals, showed that each mRNA moved to a markedly lighter polysome fraction or lost polysome association altogether in both the cytosol and the ER. GRP94, which encodes a topogenic signal, is similarly reduced in polysomes on the ER, indicating that each class of mRNA is actively translated on the ER. Combined with previous work indicating similar elongation rates for cytosolic and ER-bound ribosomes (
      • Stephens S.B.
      • Nicchitta C.V.
      Divergent regulation of protein synthesis in the cytosol and endoplasmic reticulum compartments of mammalian cells.
      ,
      • Ingolia N.T.
      • Lareau L.F.
      • Weissman J.S.
      Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes.
      ), these experiments suggest that mRNA ribosomal loading is a representative proxy for mRNA translation in each cellular compartment.
      Figure thumbnail gr2
      FIGURE 2Analysis of compartmental mRNA translational status. A–D, polyribosome profiles from the cytosol and ER in cycloheximide- (A and B) and puromycin- (C and D) treated cells are illustrated. The downward-facing arrows indicate the migration position of 80 S ribosomes. mRNA translational status was determined by [35S]Cys/Met incorporation and is depicted in the line graphs. Total RNA was isolated from gradient fractions, and the relative levels of the indicated mRNAs were determined by semiquantitative PCR.

      Ribosome Footprinting Analysis of Ribosome Loading in Cytosol and ER Compartments of HEK293 Cells

      To obtain a genome-scale survey of subcellular mRNA ribosome loading status, we utilized ribosome footprinting coupled with deep sequencing. Here, cytosolic and ER-associated polyribosomes were digested with micrococcal nuclease (
      • Arnone A.
      • Bier C.J.
      • Cotton F.A.
      • Day V.W.
      • Hazen Jr., E.E.
      • Richardson D.C.
      • Yonath A.
      • Richardson J.S.
      A high resolution structure of an inhibitor complex of the extracellular nuclease of Staphylococcus aureus. I. Experimental procedures and chain tracing.
      ,
      • Heins J.N.
      • Suriano J.R.
      • Taniuchi H.
      • Anfinsen C.B.
      Characterization of a nuclease produced by Staphylococcus aureus.
      ) to yield intact 80 S ribosomes and their associated protected mRNA fragments (
      • Wolin S.L.
      • Walter P.
      Ribosome pausing and stacking during translation of a eukaryotic mRNA.
      ). The ribosome-protected mRNA fragment complexes were isolated by ultracentrifugation and subjected to phenol/chloroform extraction, and the ∼35-nt nuclease-protected mRNA fragments were purified by acrylamide gel electrophoresis (supplemental Fig. S1). cDNA libraries were prepared using the SOLiD small RNA expression kit protocol, and the library was deeply sequenced on the SOLiD4 platform (Applied Biosystems). In parallel, total mRNA samples were prepared for deep sequencing so that the abundance of mRNAs in each compartment could be defined, allowing evaluation of ribosome footprints per mRNA, or ribosome density, for individual mRNAs.
      Deep sequencing reads representing ribosome footprints as well as total mRNA content in the cytosolic and ER compartments were mapped to RefSeq mRNAs (
      • Pruitt K.D.
      • Tatusova T.
      • Maglott D.R.
      NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts, and proteins.
      ). In Fig. 3A, the distribution of mRNAs between the cytosol and ER compartments is plotted and identifies two distinct overlapping populations: one cytosol-enriched and one ER-enriched. The overall subcellular distributions are similar to those reported previously in cDNA microarray-based studies (
      • Diehn M.
      • Eisen M.B.
      • Botstein D.
      • Brown P.O.
      Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.
      ,
      • Diehn M.
      • Bhattacharya R.
      • Botstein D.
      • Brown P.O.
      Genome-scale identification of membrane-associated human mRNAs.
      ). Notably, the cytosol-enriched mRNA population was substantially represented on the ER, a finding that mirrors earlier observations on the relative population identities of the cytosolic and ER-associated mRNAs (
      • Chen Q.
      • Jagannathan S.
      • Reid D.W.
      • Zheng T.
      • Nicchitta C.V.
      Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells.
      ,
      • Diehn M.
      • Eisen M.B.
      • Botstein D.
      • Brown P.O.
      Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.
      ,
      • Diehn M.
      • Bhattacharya R.
      • Botstein D.
      • Brown P.O.
      Genome-scale identification of membrane-associated human mRNAs.
      ,
      • Mueckler M.M.
      • Pitot H.C.
      Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations.
      ,
      • Mechler B.
      • Rabbitts T.H.
      Membrane-bound ribosomes of myeloma cells. IV. mRNA complexity of free and membrane-bound polysomes.
      ,
      • Nicchitta C.V.
      • Lerner R.S.
      • Stephens S.B.
      • Dodd R.D.
      • Pyhtila B.
      Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model.
      ). In the absence of information regarding the ribosome loading status of mRNAs in both compartments, the functional consequences of such mRNA distribution patterns are unknown. In the following, we examine this question through genome-scale analyses of ribosome loading in the cytosol and ER compartments.
      Figure thumbnail gr3
      FIGURE 3Subcellular distribution of mRNAs and translation. A, histogram distribution of the relative enrichments of mRNAs on the ER. B, histogram distribution of the fraction of translation of each mRNA occurring on the ER. C, histogram distribution of the relative ribosome loading density for each mRNA in the cytosol (cyt) and ER compartments. Also plotted in C is a moving average of the fraction of transcripts that encode a transmembrane domain or signal sequence, as predicted by TMHMM or SignalP.
      To survey the subcellular disposition of ribosome loading onto mRNAs, ribosomal footprint reads from the cytosol- and ER-derived sequencing libraries were counted to yield a measure of transcript-specific ribosome density in each compartment. As shown in Fig. 3B, the subcellular distribution of ribosome density was markedly biased to the ER compartment (Fig. 3A versus 3B), with several mRNAs loaded with ribosomes almost exclusively on the ER. Ribosome loading density, defined as the ribosome footprints per mRNA transcript, was also found to be ER-biased (Fig. 3C), indicating that most mRNAs are more densely loaded with ribosomes when associated with the ER. Of note, those mRNAs that are most efficiently loaded on the ER relative to cytosol are more likely to encode signal sequences and transmembrane domains, suggesting that the relative compartmental efficiency of translation varies for specific populations of mRNAs (Fig. 3C, inset).
      To assess the subcellular distributions of mRNAs and their compartment-specific translational status, cumulative density functions of mRNA abundance, number of ribosomes per mRNA, and ribosome loading density in the cytosol and ER compartments were determined. A broad distribution, ∼4 orders of magnitude, was observed for each parameter. The cytosol and ER diverge in the relative abundance of each metric. In particular, as a population, mRNAs are more abundant in the cytosol, indicating that this compartment serves as the primary subcellular locale for the majority of transcripts (FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4A). The number of ribosomes bound to each species of mRNA is largely similar between the two compartments, and the cytosol contains more mRNAs with relatively low quantities of associated ribosomes (Fig. 4B). The ribosome loading per mRNA molecule in each compartment indicates, on average, substantially higher loading of ribosomes on ER-bound mRNAs, which in agreement with the biochemical data presented in Fig. 1, suggests that the ER is a preferred subcellular locale for ribosome loading (Fig. 4C).
      Figure thumbnail gr4
      FIGURE 4Cumulative density plot of subcellular mRNA abundance and translation. A–C, cumulative density plots in the cytosol and ER for the abundance of each mRNA in the cytosol and ER (A), ribosomal footprints mapping to each transcript (B), and the density of ribosomes per mRNA for each gene (C).

      Compartment-specific Translational Selection of mRNAs

      We investigated the nature of the subcellular ribosome loading bias through the lens of two primary cohorts of mRNAs: those encoding cytosolic proteins (i.e. lacking topogenic signals and functioning in the cytosol or nucleus) and those whose translation products are targeted to the ER (i.e. bearing topogenic signals and targeted to cellular membranes or for secretion). By this analysis, cytosolic protein-encoding mRNAs were largely localized to the cytosol, although as noted previously, this population was ∼20% represented on the ER (Figs. 3A and 5A). Topogenic signal-encoding mRNAs were substantially ER-enriched, although with a small population displaying a cytosol enrichment (Fig. 5B). Although the global subcellular mRNA distribution is largely consistent with previous studies (
      • Chen Q.
      • Jagannathan S.
      • Reid D.W.
      • Zheng T.
      • Nicchitta C.V.
      Hierarchical regulation of mRNA partitioning between the cytoplasm and the endoplasmic reticulum of mammalian cells.
      ,
      • Diehn M.
      • Bhattacharya R.
      • Botstein D.
      • Brown P.O.
      Genome-scale identification of membrane-associated human mRNAs.
      ), the distribution of ribosome loading for both the cytosol and the ER cohorts was substantially shifted to the ER compartment. The ribosome loading of cytosolic protein-encoding mRNAs displayed a broad distribution, with the peak gene density occurring at ∼45% fractional translation on the ER (Fig. 5C). With but few exceptions, mRNAs encoding ER-targeted proteins were loaded with ribosomes almost exclusively on the ER (Fig. 5D).
      Figure thumbnail gr5
      FIGURE 5Subcellular mRNA partitioning and ribosome loading patterns of different mRNA cohorts. A–D, histogram distributions of the fraction of ER-associated mRNAs for cytosolic protein-encoding (A) and topogenic signal-encoding mRNAs (B) and the fraction of ribosome loading on the ER for cytosolic protein-encoding (C) and topogenic signal-encoding (D) mRNAs.
      To examine compartment-specific properties of translation in further detail, mRNA abundance and ribosome density for these two gene categories in the cytosol and ER were examined (Fig. 6, A and B). In the cytosol, mRNAs encoding ER-targeted proteins are substantially less abundant and are sparsely loaded with ribosomes, indicating that translation is significantly less robust for this cohort in the cytosol than for their counterparts encoding cytosol-targeted proteins. In contrast, mRNAs encoding both cytosol-targeted and ER-targeted proteins are similarly abundant and loaded with ribosomes when associated with the ER, suggesting that ribosomes in this compartment contribute to the synthesis of nearly all cellular proteins. This pattern is reinforced in the plot depicted in Fig. 6C, where ribosome loading densities for these two cohorts are similar on the ER, but divergent in the cytosol. A strong positive correlation between ribosome loading in the two compartments for mRNAs encoding cytosolic proteins was observed, suggesting that the translational regulatory machinery may be shared. This correlation is substantially weaker for mRNAs encoding ER-targeted proteins. We also examined the relationship between mRNA localization and the localization of ribosome loading and observed that for mRNAs encoding cytosolic proteins, there is no substantial correlation between the two variables, suggesting that the localizations of mRNAs and their translation are likely under distinct regulation (Fig. 6D). In contrast, there is a significant positive correlation for mRNAs encoding ER-targeted proteins. Each of the divergences discussed here were statistically significant (supplemental Table S2).
      Figure thumbnail gr6
      FIGURE 6Divergent patterns of subcellular ribosome loading for different mRNA cohorts. A and B, the abundance of each mRNA plotted against its loading with ribosomes in the cytosol (A) and ER (B). C, the density of ribosomal reads per mRNA in the cytosol plotted against that in the ER. D, the fraction of associated ribosomes for each mRNA on the ER plotted against the fraction of that mRNA present on the ER. In each plot, mRNAs encoding proteins targeted to the cytosol and ER are colored separately. Ellipses represent a 50th percentile density, and points represent individual mRNAs.
      To characterize the nature of cytosolic protein-encoding mRNAs that are preferentially loaded with ribosomes in the cytosol or the ER, the enrichment of GOs was examined as a function of ribosome loading density in either compartment. The analysis controls for the relative abundance of mRNAs in each compartment and so is only sensitive to differences in ribosome loading. Identified GOs are noted in Table 1; the relative enrichment for all GOs is provided in supplemental Table S3. The cohort of mRNAs encoding cytosolic products that were more heavily loaded with ribosomes on the ER was enriched for regulatory and dynamic cell functions, particularly the cell cycle. For example, the cell cycle regulators p53 (
      • Matlashewski G.
      • Lamb P.
      • Pim D.
      • Peacock J.
      • Crawford L.
      • Benchimol S.
      Isolation and characterization of a human p53 cDNA clone: expression of the human p53 gene.
      ) and Myc (
      • He T.C.
      • Sparks A.B.
      • Rago C.
      • Hermeking H.
      • Zawel L.
      • da Costa L.T.
      • Morin P.J.
      • Vogelstein B.
      • Kinzler K.W.
      Identification of c-MYC as a target of the APC pathway.
      ) were both 4.5-fold more heavily loaded with ribosomes on the ER when compared with the cytosol.
      TABLE 1Selected compartmental gene ontology enrichments
      Gene ontologyp value
      ER-enriched translation
      Kinase regulator activity0.0020
      Cell cycle arrest0.0065
      System development0.0095
      mRNA metabolic process0.0180
      Cytosol-enriched translation
      Phosphoinositide binding0.0065
      Receptor protein signaling pathway0.0080
      Kinase activity0.0195
      Cellular lipid metabolic process0.0245
      Several of the GO-defined cohorts that were efficiently translated in the cytosol were found to be associated with biochemical functions of relevance to plasma membrane function. For example, mRNAs encoding intestinal cell kinase (ICK), a protein kinase that localizes to basal membranes of epithelia (
      • Togawa K.
      • Yan Y.X.
      • Inomoto T.
      • Slaugenhaupt S.
      • Rustgi A.K.
      Intestinal cell kinase (ICK) localizes to the crypt region and requires a dual phosphorylation site found in map kinases.
      ), are 4-fold more ribosome-loaded in the cytosol. These GOs may represent instances of mRNAs that are translationally activated in particular regions of the cell where their protein products are functional, in this case the areas proximal to the plasma membrane, which is consistent with current views on coupled mRNA localization and translational regulation (
      • Martin K.C.
      • Ephrussi A.
      mRNA localization: gene expression in the spatial dimension.
      ).

      ER and Cytosol Diverge in Their Spatial Patterns of Ribosome Loading

      In light of the distinct patterns of compartment-enriched ribosome loading noted above and their potential repercussions regarding localized translation, we analyzed the ribosome mapping dataset to derive insight into stage- (initiation, elongation, and termination) specific translational regulation and how this might diverge between the cytosol and ER compartments. This analysis assumes that read density is a representation of occupancy time and therefore the relative kinetics at each position. Total read density was plotted relative to the start and stop codon in the cytosolic (Fig. 7A) and ER-associated mRNAs (Fig. 7B). Both compartments display patterns in which ribosome density increases at the start codon and decreases at the stop codon, consistent with previous ribosome profiling studies (
      • Ingolia N.T.
      • Ghaemmaghami S.
      • Newman J.R.
      • Weissman J.S.
      Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.
      ,
      • Guo H.
      • Ingolia N.T.
      • Weissman J.S.
      • Bartel D.P.
      Mammalian microRNAs predominantly act to decrease target mRNA levels.
      ). Also apparent is a three-base periodicity to the density, indicating that the single-codon procession of the ribosome was accurately captured. The cytosolic compartment displayed a clear density enrichment near the stop codon, suggesting that termination may be slower in the cytosol relative to the ER. The processivity of elongation was calculated by monitoring the density of ribosomes as a function of coding sequence position (supplemental Fig. S4). This analysis suggested that translation in the cytosol was less processive than that in the ER (Fig. 8), with decay constants of 0.00048 and 0.00019, respectively. These constants correspond to an average ribosome translational lifetime of 2083 amino acids in the cytosol and 5268 amino acids on the ER. We hypothesized that this disparity could lead to enrichment of translation of longer mRNAs on the ER, but no such trend was apparent. Together, these analyses suggest that the composite biochemical reactions of translation in the cytosol and ER possess kinetic differences.
      Figure thumbnail gr7
      FIGURE 7Spatial patterns of ribosome occupancy on mRNAs. A and B, density of ribosomes near the start and stop codon for cytosolic (A) and ER (B) compartments.
      Figure thumbnail gr8
      FIGURE 8Ribosome processivity differs between cytosolic (Cyt) and ER-bound ribosomes. Ribosome processivity was calculated by fitting an exponential decay curve to the ribosome density in each compartment. Error bars represent at 95% confidence interval.

      DISCUSSION

      In the current study, we have used ribosome profiling to investigate the in vivo role of the ER in mRNA translation and report two primary observations: 1) mRNAs encoding cytosolic proteins were broadly represented in the ER ribosome-associated mRNA pool and 2) steady-state ribosome loading on ER-bound mRNAs was substantially higher than in the cytosol. In demonstrating that ER-localized mRNAs display higher ribosome loading than their cytosolic counterparts, these findings expand the landscape of post-transcriptional regulation of gene expression to include the partitioning of mRNAs between the cytosol and ER compartments.
      A finding of particular interest in the present study was the observed discordance between mRNA translational status and subcellular localization, indicating that mRNA localization and translational activity can comprise two distinctly regulated processes. The importance of this finding is underscored by the enhanced ribosome loading for ER-associated cytosolic/nucleoplasmic protein-encoding mRNAs encoding key regulatory proteins and suggests that the ER may serve as a preferred locale for the synthesis of proteins involved in, for example, the cell cycle and gene expression. When viewed with respect to previous studies demonstrating both an enhanced half-life for ER-associated mRNAs (
      • Hyde M.
      • Block-Alper L.
      • Felix J.
      • Webster P.
      • Meyer D.I.
      Induction of secretory pathway components in yeast is associated with increased stability of their mRNA.
      ) and the finding that ER-associated mRNAs are excluded from the stress granule-directed trafficking pathways (
      • Unsworth H.
      • Raguz S.
      • Edwards H.J.
      • Higgins C.F.
      • Yagüe E.
      mRNA escape from stress granule sequestration is dictated by localization to the endoplasmic reticulum.
      ), the sum of findings to date points toward a distinct, broadly significant role for the ER in the expression of the mRNA transcriptome. We do emphasize, however, that the ribosome footprinting/RNA-Seq analysis is a proxy assay. If, for example, a given mRNA contains abundant pause sites and/or, as noted above, is subject to compartment-specific translational regulation, an enhanced ribosome loading density could reflect a phenomenon other than strict steady-state translational status. These scenarios are under current investigation.
      At present, the molecular basis for the enhanced ribosome loading status of ER-associated mRNAs is unknown. One hypothesis for the divergent translational efficiencies of the two compartments is that RNA-binding proteins, many of which modulate a wide range of post-transcriptional processes (
      • Mansfield K.D.
      • Keene J.D.
      The ribonome: a dominant force in coordinating gene expression.
      ) and confer compartment-specific translational efficiency (
      • Besse F.
      • Ephrussi A.
      Translational control of localized mRNAs: restricting protein synthesis in space and time.
      ), are compartmentally segregated between cytosolic and ER-bound polysomes. Indeed, it is plausible that one or more of the many RNA-binding proteins shown to change the translational status of mRNAs operate by modulating translation in a compartment-specific manner. A related mechanism was recently described in the case of RPL38, a ribosomal protein that promotes translation of a specific subset of Hox genes (
      • Kondrashov N.
      • Pusic A.
      • Stumpf C.R.
      • Shimizu K.
      • Hsieh A.C.
      • Xue S.
      • Ishijima J.
      • Shiroishi T.
      • Barna M.
      Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning.
      ).
      Interestingly, despite ribosome footprinting indicating a far greater average ribosome density in the ER, the cytosol and ER polyribosome profiles, as assessed by sucrose gradient fractionation, are similar. This divergence indicates that there may exist a substantial pool of mRNAs that are not ribosome-associated and that this pool is largely segregated to the cytosol. Indeed, it has been demonstrated that ∼25% of all yeast mRNAs are not ribosome-associated (
      • Arava Y.
      • Wang Y.
      • Storey J.D.
      • Liu C.L.
      • Brown P.O.
      • Herschlag D.
      Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae.
      ). Should these mRNAs be predominantly localized to the cytosol, perhaps associated with processing bodies or stress granules, this would indicate a similar ribosome loading density in each compartment for those mRNAs that are available for translation. Given, as noted above, that ER-bound mRNAs are resistant to recruitment into stress granules (
      • Unsworth H.
      • Raguz S.
      • Edwards H.J.
      • Higgins C.F.
      • Yagüe E.
      mRNA escape from stress granule sequestration is dictated by localization to the endoplasmic reticulum.
      ), it is plausible that compartmental differences in stress granule and processing body association lead to a large population of cytosol-localized mRNAs that are not associated with polyribosomes. This model is consistent with Fig. 2A, where mRNAs in the cytosol are more frequently not associated with polysomes than those on the ER. The ER may therefore represent a cellular compartment for dedicated protein synthesis, with the cytosol compartment engaged in the ancillary functions of mRNA storage, degradation, and related processing.
      This study introduces the concept of a subcellular architecture to mRNA localization and translation. Although the functional ramifications and mechanistic details remain to be developed, we suggest that the partitioning of the mRNA transcriptome between the cytosol and ER may represent a novel post-transcriptional regulation mechanism with broad consequences for the expression of the cellular proteome (
      • Nicchitta C.V.
      • Lerner R.S.
      • Stephens S.B.
      • Dodd R.D.
      • Pyhtila B.
      Pathways for compartmentalizing protein synthesis in eukaryotic cells: the template-partitioning model.
      ).

      Acknowledgments

      We are grateful for the helpful critiques provided by members of the Nicchitta laboratory, the laboratories of Uwe Ohler and Jack Keene, and our colleagues Zackary Scholl and Charles Cooper. We thank Nicholas Hoang and Lisa Bukovnik of the Duke University Genome Sequencing & Analysis Core Resource for transcriptome sequencing support and Sujatha Jagannathan for providing key advice and supporting data on cell fractionation.

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