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p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum.

Cui XA, Zhang H, Palazzo AF - PLoS Biol. (2012)

Bottom Line: Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation.In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER.It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
In metazoans, the majority of mRNAs coding for secreted and membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). Although the targeting of these transcripts to the surface of the ER can be mediated by the translation of a signal sequence and their maintenance is mediated by interactions between the ribosome and the translocon, it is becoming increasingly clear that additional ER-localization pathways exist. Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation. Using a mass spectrometry analysis of proteins that associate with ER-bound polysomes, we identified putative mRNA receptors that may mediate this alternative mechanism, including p180, an abundant, positively charged membrane-bound protein. We demonstrate that p180 over-expression can enhance the association of generic mRNAs with the ER. We then show that p180 contains a lysine-rich region that can directly interact with RNA in vitro. Finally, we demonstrate that p180 is required for the efficient ER-anchoring of bulk poly(A) and of certain transcripts, such as placental alkaline phosphatase and calreticulin, to the ER. In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER. It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.

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ALPP and CALR, but not t-ftz or INSL3, mRNA remain associated with the ER independently of ribosomes and translation.(A–E) COS-7 cells were transfected with plasmids containing either the t-ftz (A), INSL3 (A–B), ALPP (A, C), cyto-ALPP (a version of ALPP lacking signal sequence and transmembrane domain coding regions; A, D–E), or CALR (A) genes and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (“Cont”), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels B–D for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused INSL3 mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing cyto-ALPP mRNA. cyto-ALPP mRNA was visualized by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of cyto-ALPP mRNA (red) and Trapα (green) in the overlay. All scale bars = 20 µm.
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pbio-1001336-g002: ALPP and CALR, but not t-ftz or INSL3, mRNA remain associated with the ER independently of ribosomes and translation.(A–E) COS-7 cells were transfected with plasmids containing either the t-ftz (A), INSL3 (A–B), ALPP (A, C), cyto-ALPP (a version of ALPP lacking signal sequence and transmembrane domain coding regions; A, D–E), or CALR (A) genes and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (“Cont”), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels B–D for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused INSL3 mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing cyto-ALPP mRNA. cyto-ALPP mRNA was visualized by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of cyto-ALPP mRNA (red) and Trapα (green) in the overlay. All scale bars = 20 µm.

Mentions: Next, the distribution of transcripts from individual genes was monitored by conventional FISH in COS-7 cells. The majority of these genes have a signal sequence coding region (SSCR), which not only encodes ER-targeting polypeptides but also contains an RNA element that promotes nuclear export and the proper cytoplasmic localization of transcripts [30],[37]. With this in mind, we first investigated the cellular distribution of the reporter transcript t-ftz, which contains the SSCR from a mouse Major Histocompatibility Complex (MHC) H2kb gene [30]. Besides the SSCR, this artificial transcript does not contain any sequence that is normally associated with the ER, as it was derived from a transcription factor gene from Drosophila[38]. Interestingly, t-ftz mRNA, which localizes to the ER in extracted cells (Figure S1A), no longer associated with this organelle after ribosome disruption using either HHT or puromycin/EDTA (Figure 2A, Figure S2A). Note that the amount of nuclear t-ftz transcript was unaltered by any of the treatments (Figure 2A), indicating that the change in ER-associated fluorescence was not due to changes in expression levels or FISH efficiency. Thus, we conclude that although the MHC SSCR can promote nuclear export, it is not sufficient to allow mRNAs to be maintained on the surface of the ER after ribosome dissociation.


p180 promotes the ribosome-independent localization of a subset of mRNA to the endoplasmic reticulum.

Cui XA, Zhang H, Palazzo AF - PLoS Biol. (2012)

ALPP and CALR, but not t-ftz or INSL3, mRNA remain associated with the ER independently of ribosomes and translation.(A–E) COS-7 cells were transfected with plasmids containing either the t-ftz (A), INSL3 (A–B), ALPP (A, C), cyto-ALPP (a version of ALPP lacking signal sequence and transmembrane domain coding regions; A, D–E), or CALR (A) genes and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (“Cont”), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels B–D for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused INSL3 mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing cyto-ALPP mRNA. cyto-ALPP mRNA was visualized by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of cyto-ALPP mRNA (red) and Trapα (green) in the overlay. All scale bars = 20 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362647&req=5

pbio-1001336-g002: ALPP and CALR, but not t-ftz or INSL3, mRNA remain associated with the ER independently of ribosomes and translation.(A–E) COS-7 cells were transfected with plasmids containing either the t-ftz (A), INSL3 (A–B), ALPP (A, C), cyto-ALPP (a version of ALPP lacking signal sequence and transmembrane domain coding regions; A, D–E), or CALR (A) genes and allowed to express mRNA for 18–24 h. The cells were then treated with DMSO (“Cont”), puromycin, or HHT for 30 min, and then extracted with digitonin alone or with 20 mM EDTA. Cells were then fixed, stained for mRNA using specific FISH probes, and imaged (see panels B–D for examples). The fluorescence intensities of mRNA in the ER and nucleus in the micrographs were quantified (A). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. Note that although ribosome disruption caused INSL3 mRNA to dissociate from the ER, the nuclear mRNA was unaffected (B, nuclei are denoted by arrows). (E) A single field of view containing a single HHT-treated, digitonin-extracted, COS-7 cell expressing cyto-ALPP mRNA. cyto-ALPP mRNA was visualized by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of cyto-ALPP mRNA (red) and Trapα (green) in the overlay. All scale bars = 20 µm.
Mentions: Next, the distribution of transcripts from individual genes was monitored by conventional FISH in COS-7 cells. The majority of these genes have a signal sequence coding region (SSCR), which not only encodes ER-targeting polypeptides but also contains an RNA element that promotes nuclear export and the proper cytoplasmic localization of transcripts [30],[37]. With this in mind, we first investigated the cellular distribution of the reporter transcript t-ftz, which contains the SSCR from a mouse Major Histocompatibility Complex (MHC) H2kb gene [30]. Besides the SSCR, this artificial transcript does not contain any sequence that is normally associated with the ER, as it was derived from a transcription factor gene from Drosophila[38]. Interestingly, t-ftz mRNA, which localizes to the ER in extracted cells (Figure S1A), no longer associated with this organelle after ribosome disruption using either HHT or puromycin/EDTA (Figure 2A, Figure S2A). Note that the amount of nuclear t-ftz transcript was unaltered by any of the treatments (Figure 2A), indicating that the change in ER-associated fluorescence was not due to changes in expression levels or FISH efficiency. Thus, we conclude that although the MHC SSCR can promote nuclear export, it is not sufficient to allow mRNAs to be maintained on the surface of the ER after ribosome dissociation.

Bottom Line: Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation.In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER.It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
In metazoans, the majority of mRNAs coding for secreted and membrane-bound proteins are translated on the surface of the endoplasmic reticulum (ER). Although the targeting of these transcripts to the surface of the ER can be mediated by the translation of a signal sequence and their maintenance is mediated by interactions between the ribosome and the translocon, it is becoming increasingly clear that additional ER-localization pathways exist. Here we demonstrate that many of these mRNAs can be targeted to, and remain associated with, the ER independently of ribosomes and translation. Using a mass spectrometry analysis of proteins that associate with ER-bound polysomes, we identified putative mRNA receptors that may mediate this alternative mechanism, including p180, an abundant, positively charged membrane-bound protein. We demonstrate that p180 over-expression can enhance the association of generic mRNAs with the ER. We then show that p180 contains a lysine-rich region that can directly interact with RNA in vitro. Finally, we demonstrate that p180 is required for the efficient ER-anchoring of bulk poly(A) and of certain transcripts, such as placental alkaline phosphatase and calreticulin, to the ER. In summary, we provide, to our knowledge, the first mechanistic details for an alternative pathway to target and maintain mRNA at the ER. It is likely that this alternative pathway not only enhances the fidelity of protein sorting, but also localizes mRNAs to various subdomains of the ER and thus contributes to cellular organization.

Show MeSH
Related in: MedlinePlus