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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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The initial ER-targeting of ALPP and CALR, but not t-ftz or INSL3, mRNA occurs independently of translation or ribosomes.(A–B) COS-7 cells were pretreated with DMSO (“Control”) or HHT for 15 min, then microinjected with plasmids containing either the ALPP, INSL3, t-ftz, or CALR genes and allowed to express mRNA for 2 h in the presence of DMSO or HHT. To label the microinjected cells, Alexa488-conjugated 70 kD dextran was co-injected (see insets in A). The cells were then extracted with digitonin, fixed, stained for mRNA using specific FISH probes, and imaged (A). The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified (B). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C) COS-7 cells were pretreated with HHT for 15 min, then microinjected with plasmids containing the ALPP gene. Cells were then incubated for 2 h in the presence of HHT, then extracted with digitonin, fixed, and then co-stained for ALPP mRNA by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of ALPP mRNA (red) and Trapα (green). All scale bars = 20 µm.
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pbio-1001336-g004: The initial ER-targeting of ALPP and CALR, but not t-ftz or INSL3, mRNA occurs independently of translation or ribosomes.(A–B) COS-7 cells were pretreated with DMSO (“Control”) or HHT for 15 min, then microinjected with plasmids containing either the ALPP, INSL3, t-ftz, or CALR genes and allowed to express mRNA for 2 h in the presence of DMSO or HHT. To label the microinjected cells, Alexa488-conjugated 70 kD dextran was co-injected (see insets in A). The cells were then extracted with digitonin, fixed, stained for mRNA using specific FISH probes, and imaged (A). The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified (B). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C) COS-7 cells were pretreated with HHT for 15 min, then microinjected with plasmids containing the ALPP gene. Cells were then incubated for 2 h in the presence of HHT, then extracted with digitonin, fixed, and then co-stained for ALPP mRNA by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of ALPP mRNA (red) and Trapα (green). All scale bars = 20 µm.

Mentions: Our data indicated that once certain transcripts are targeted to the ER, they are retained on the surface of this organelle independently of ribosomes. It, however, remained unclear whether the initial targeting step could occur independently of translation. To address this question, cells were pretreated with HHT to inhibit translation and then microinjected with plasmid DNA. Two hours later, the distribution of the newly synthesized mRNA, which was never in contact with functional ribosomes, was assessed. Surprisingly, both ALPP and CALR mRNA targeted to the ER independently of translation (Figure 4A–B). In contrast INSL3 mRNA only displayed weak translation-independent targeting activity, while t-ftz mRNA failed to target to the ER under these conditions (Figure 4A–B). All of the tested transcripts targeted to the ER in the absence of translation inhibitors (i.e., DMSO treatment). To ensure that any changes in fluorescence were not due to changes in mRNA expression or variability in FISH staining, we monitored the nuclear mRNA levels of each construct, and these did not drastically change between experiments (Figure 4B). The targeting of ALPP mRNA to the ER in HHT-treated cells was confirmed by co-localization of the digitonin-resistant transcripts with Trapα (Figure 4C).


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

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

The initial ER-targeting of ALPP and CALR, but not t-ftz or INSL3, mRNA occurs independently of translation or ribosomes.(A–B) COS-7 cells were pretreated with DMSO (“Control”) or HHT for 15 min, then microinjected with plasmids containing either the ALPP, INSL3, t-ftz, or CALR genes and allowed to express mRNA for 2 h in the presence of DMSO or HHT. To label the microinjected cells, Alexa488-conjugated 70 kD dextran was co-injected (see insets in A). The cells were then extracted with digitonin, fixed, stained for mRNA using specific FISH probes, and imaged (A). The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified (B). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C) COS-7 cells were pretreated with HHT for 15 min, then microinjected with plasmids containing the ALPP gene. Cells were then incubated for 2 h in the presence of HHT, then extracted with digitonin, fixed, and then co-stained for ALPP mRNA by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of ALPP mRNA (red) and Trapα (green). All scale bars = 20 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362647&req=5

pbio-1001336-g004: The initial ER-targeting of ALPP and CALR, but not t-ftz or INSL3, mRNA occurs independently of translation or ribosomes.(A–B) COS-7 cells were pretreated with DMSO (“Control”) or HHT for 15 min, then microinjected with plasmids containing either the ALPP, INSL3, t-ftz, or CALR genes and allowed to express mRNA for 2 h in the presence of DMSO or HHT. To label the microinjected cells, Alexa488-conjugated 70 kD dextran was co-injected (see insets in A). The cells were then extracted with digitonin, fixed, stained for mRNA using specific FISH probes, and imaged (A). The fluorescence intensity of mRNA in the ER and nucleus in the micrographs were quantified (B). Each bar represents the average and standard error of three independent experiments, each consisting of the average integrated intensity of 30 cells over background. (C) COS-7 cells were pretreated with HHT for 15 min, then microinjected with plasmids containing the ALPP gene. Cells were then incubated for 2 h in the presence of HHT, then extracted with digitonin, fixed, and then co-stained for ALPP mRNA by FISH and for Trapα protein by immunofluorescence. Note the extensive co-localization of ALPP mRNA (red) and Trapα (green). All scale bars = 20 µm.
Mentions: Our data indicated that once certain transcripts are targeted to the ER, they are retained on the surface of this organelle independently of ribosomes. It, however, remained unclear whether the initial targeting step could occur independently of translation. To address this question, cells were pretreated with HHT to inhibit translation and then microinjected with plasmid DNA. Two hours later, the distribution of the newly synthesized mRNA, which was never in contact with functional ribosomes, was assessed. Surprisingly, both ALPP and CALR mRNA targeted to the ER independently of translation (Figure 4A–B). In contrast INSL3 mRNA only displayed weak translation-independent targeting activity, while t-ftz mRNA failed to target to the ER under these conditions (Figure 4A–B). All of the tested transcripts targeted to the ER in the absence of translation inhibitors (i.e., DMSO treatment). To ensure that any changes in fluorescence were not due to changes in mRNA expression or variability in FISH staining, we monitored the nuclear mRNA levels of each construct, and these did not drastically change between experiments (Figure 4B). The targeting of ALPP mRNA to the ER in HHT-treated cells was confirmed by co-localization of the digitonin-resistant transcripts with Trapα (Figure 4C).

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