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Alternative 3' UTRs act as scaffolds to regulate membrane protein localization.

Berkovits BD, Mayr C - Nature (2015)

Bottom Line: This facilitates interaction of SET with the newly translated cytoplasmic domains of CD47 and results in subsequent translocation of CD47 to the plasma membrane via activated RAC1 (ref. 5).Thus, ApA contributes to the functional diversity of the proteome without changing the amino acid sequence. 3' UTR-dependent protein localization has the potential to be a widespread trafficking mechanism for membrane proteins because HuR binds to thousands of mRNAs, and we show that the long 3' UTRs of CD44, ITGA1 and TNFRSF13C, which are bound by HuR, increase surface protein expression compared to their corresponding short 3' UTRs.We propose that during translation the scaffold function of 3' UTRs facilitates binding of proteins to nascent proteins to direct their transport or function--and this role of 3' UTRs can be regulated by ApA.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, 1275 York Ave, New York, New York 10065, USA.

ABSTRACT
About half of human genes use alternative cleavage and polyadenylation (ApA) to generate messenger RNA transcripts that differ in the length of their 3' untranslated regions (3' UTRs) while producing the same protein. Here we show in human cell lines that alternative 3' UTRs differentially regulate the localization of membrane proteins. The long 3' UTR of CD47 enables efficient cell surface expression of CD47 protein, whereas the short 3' UTR primarily localizes CD47 protein to the endoplasmic reticulum. CD47 protein localization occurs post-translationally and independently of RNA localization. In our model of 3' UTR-dependent protein localization, the long 3' UTR of CD47 acts as a scaffold to recruit a protein complex containing the RNA-binding protein HuR (also known as ELAVL1) and SET to the site of translation. This facilitates interaction of SET with the newly translated cytoplasmic domains of CD47 and results in subsequent translocation of CD47 to the plasma membrane via activated RAC1 (ref. 5). We also show that CD47 protein has different functions depending on whether it was generated by the short or long 3' UTR isoforms. Thus, ApA contributes to the functional diversity of the proteome without changing the amino acid sequence. 3' UTR-dependent protein localization has the potential to be a widespread trafficking mechanism for membrane proteins because HuR binds to thousands of mRNAs, and we show that the long 3' UTRs of CD44, ITGA1 and TNFRSF13C, which are bound by HuR, increase surface protein expression compared to their corresponding short 3' UTRs. We propose that during translation the scaffold function of 3' UTRs facilitates binding of proteins to nascent proteins to direct their transport or function--and this role of 3' UTRs can be regulated by ApA.

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CD47 protein has different functions dependent on whether it was generated by the SU or LU isoform(A) To generate GFP-CD47, GFP was inserted in frame between the signal peptide and the rest of the CD47 open reading frame. GFP-CD47 was fused with either the long or short CD47 3'UTR, called CD47-LU and CD47-SU respectively.(B) FACS analysis of surface (surf.; light blue) and total (dark blue) GFP–CD47 expression in transfected JinB8 cells. Shown as in Fig. 1g. Representative images from four experiments.(C) FACS analysis of GFP expression after transfection of CD47-LU or CD47-SU with or without co-transfection of dominant-negative RAC1 (N17RAC1). Shown as in Fig. 1g. Representative images from n = 7 (LU) and n = 2 (SU) experiments.(D) Fraction of Mitomycin C-treated cells that survived at d3 after co-culture with macrophages is displayed for Jurkat, JinB8 (CD47−/−) and the GFP+ JinB8 cells after nucleofection of CD47-LU or CD47-SU. Shown is mean ± s.d., n = 3 biological replicates. **P < 0.005, *P < 0.02, NS, not significant (P > 0.05), two-sided t-test for independent samples.(E) The fraction of surviving cells (TO-PRO3 negative) measured by FACS analysis at day 3 after γ-irradiation is shown for the same populations as in (d). Shown is mean ± s.d., n = 3 biological replicates of the 20% of cells with the highest GFP expression. Gy, Gray.(F) Fluorescence confocal microscopy of permeabilized U251 cells after transfection of CD47-LU or CD47-SU co-stained with anti-RAC1 antibody. Yellow indicates co-localization. Representative images from hundreds of cells. Scale bars, 10 µm.(G) Immunoprecipitation of endogenous RAC1–GTP (active RAC1) in HEK293 cells after transfection of CD47-LU, CD47-SU, or empty vector. Total RAC1 and GFP–CD47 were measured from input. n=3 biological replicates.
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Figure 4: CD47 protein has different functions dependent on whether it was generated by the SU or LU isoform(A) To generate GFP-CD47, GFP was inserted in frame between the signal peptide and the rest of the CD47 open reading frame. GFP-CD47 was fused with either the long or short CD47 3'UTR, called CD47-LU and CD47-SU respectively.(B) FACS analysis of surface (surf.; light blue) and total (dark blue) GFP–CD47 expression in transfected JinB8 cells. Shown as in Fig. 1g. Representative images from four experiments.(C) FACS analysis of GFP expression after transfection of CD47-LU or CD47-SU with or without co-transfection of dominant-negative RAC1 (N17RAC1). Shown as in Fig. 1g. Representative images from n = 7 (LU) and n = 2 (SU) experiments.(D) Fraction of Mitomycin C-treated cells that survived at d3 after co-culture with macrophages is displayed for Jurkat, JinB8 (CD47−/−) and the GFP+ JinB8 cells after nucleofection of CD47-LU or CD47-SU. Shown is mean ± s.d., n = 3 biological replicates. **P < 0.005, *P < 0.02, NS, not significant (P > 0.05), two-sided t-test for independent samples.(E) The fraction of surviving cells (TO-PRO3 negative) measured by FACS analysis at day 3 after γ-irradiation is shown for the same populations as in (d). Shown is mean ± s.d., n = 3 biological replicates of the 20% of cells with the highest GFP expression. Gy, Gray.(F) Fluorescence confocal microscopy of permeabilized U251 cells after transfection of CD47-LU or CD47-SU co-stained with anti-RAC1 antibody. Yellow indicates co-localization. Representative images from hundreds of cells. Scale bars, 10 µm.(G) Immunoprecipitation of endogenous RAC1–GTP (active RAC1) in HEK293 cells after transfection of CD47-LU, CD47-SU, or empty vector. Total RAC1 and GFP–CD47 were measured from input. n=3 biological replicates.

Mentions: To test if the difference in surface localization has phenotypic consequences, we added the ECD of CD47 to the GFP constructs (called CD47-LU or CD47-SU; Fig. 4a). Both constructs resulted in comparable overall CD47 protein levels (Extended Data Fig. 7a). CD47-LU efficiently localized to the cell surface via UDPL mediated by active RAC1 (Fig. 4b, c and Extended Data Fig. 7b). Whereas GFP expressed from the GFP-TM-SU construct nearly completely localized to the endoplasmic reticulum (Fig. 1f–h), CD47-SU primarily localizes to the endoplasmic reticulum, but also localizes partially to the cell surface, but independently of active RAC1 (Fig. 4b, c).


Alternative 3' UTRs act as scaffolds to regulate membrane protein localization.

Berkovits BD, Mayr C - Nature (2015)

CD47 protein has different functions dependent on whether it was generated by the SU or LU isoform(A) To generate GFP-CD47, GFP was inserted in frame between the signal peptide and the rest of the CD47 open reading frame. GFP-CD47 was fused with either the long or short CD47 3'UTR, called CD47-LU and CD47-SU respectively.(B) FACS analysis of surface (surf.; light blue) and total (dark blue) GFP–CD47 expression in transfected JinB8 cells. Shown as in Fig. 1g. Representative images from four experiments.(C) FACS analysis of GFP expression after transfection of CD47-LU or CD47-SU with or without co-transfection of dominant-negative RAC1 (N17RAC1). Shown as in Fig. 1g. Representative images from n = 7 (LU) and n = 2 (SU) experiments.(D) Fraction of Mitomycin C-treated cells that survived at d3 after co-culture with macrophages is displayed for Jurkat, JinB8 (CD47−/−) and the GFP+ JinB8 cells after nucleofection of CD47-LU or CD47-SU. Shown is mean ± s.d., n = 3 biological replicates. **P < 0.005, *P < 0.02, NS, not significant (P > 0.05), two-sided t-test for independent samples.(E) The fraction of surviving cells (TO-PRO3 negative) measured by FACS analysis at day 3 after γ-irradiation is shown for the same populations as in (d). Shown is mean ± s.d., n = 3 biological replicates of the 20% of cells with the highest GFP expression. Gy, Gray.(F) Fluorescence confocal microscopy of permeabilized U251 cells after transfection of CD47-LU or CD47-SU co-stained with anti-RAC1 antibody. Yellow indicates co-localization. Representative images from hundreds of cells. Scale bars, 10 µm.(G) Immunoprecipitation of endogenous RAC1–GTP (active RAC1) in HEK293 cells after transfection of CD47-LU, CD47-SU, or empty vector. Total RAC1 and GFP–CD47 were measured from input. n=3 biological replicates.
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Figure 4: CD47 protein has different functions dependent on whether it was generated by the SU or LU isoform(A) To generate GFP-CD47, GFP was inserted in frame between the signal peptide and the rest of the CD47 open reading frame. GFP-CD47 was fused with either the long or short CD47 3'UTR, called CD47-LU and CD47-SU respectively.(B) FACS analysis of surface (surf.; light blue) and total (dark blue) GFP–CD47 expression in transfected JinB8 cells. Shown as in Fig. 1g. Representative images from four experiments.(C) FACS analysis of GFP expression after transfection of CD47-LU or CD47-SU with or without co-transfection of dominant-negative RAC1 (N17RAC1). Shown as in Fig. 1g. Representative images from n = 7 (LU) and n = 2 (SU) experiments.(D) Fraction of Mitomycin C-treated cells that survived at d3 after co-culture with macrophages is displayed for Jurkat, JinB8 (CD47−/−) and the GFP+ JinB8 cells after nucleofection of CD47-LU or CD47-SU. Shown is mean ± s.d., n = 3 biological replicates. **P < 0.005, *P < 0.02, NS, not significant (P > 0.05), two-sided t-test for independent samples.(E) The fraction of surviving cells (TO-PRO3 negative) measured by FACS analysis at day 3 after γ-irradiation is shown for the same populations as in (d). Shown is mean ± s.d., n = 3 biological replicates of the 20% of cells with the highest GFP expression. Gy, Gray.(F) Fluorescence confocal microscopy of permeabilized U251 cells after transfection of CD47-LU or CD47-SU co-stained with anti-RAC1 antibody. Yellow indicates co-localization. Representative images from hundreds of cells. Scale bars, 10 µm.(G) Immunoprecipitation of endogenous RAC1–GTP (active RAC1) in HEK293 cells after transfection of CD47-LU, CD47-SU, or empty vector. Total RAC1 and GFP–CD47 were measured from input. n=3 biological replicates.
Mentions: To test if the difference in surface localization has phenotypic consequences, we added the ECD of CD47 to the GFP constructs (called CD47-LU or CD47-SU; Fig. 4a). Both constructs resulted in comparable overall CD47 protein levels (Extended Data Fig. 7a). CD47-LU efficiently localized to the cell surface via UDPL mediated by active RAC1 (Fig. 4b, c and Extended Data Fig. 7b). Whereas GFP expressed from the GFP-TM-SU construct nearly completely localized to the endoplasmic reticulum (Fig. 1f–h), CD47-SU primarily localizes to the endoplasmic reticulum, but also localizes partially to the cell surface, but independently of active RAC1 (Fig. 4b, c).

Bottom Line: This facilitates interaction of SET with the newly translated cytoplasmic domains of CD47 and results in subsequent translocation of CD47 to the plasma membrane via activated RAC1 (ref. 5).Thus, ApA contributes to the functional diversity of the proteome without changing the amino acid sequence. 3' UTR-dependent protein localization has the potential to be a widespread trafficking mechanism for membrane proteins because HuR binds to thousands of mRNAs, and we show that the long 3' UTRs of CD44, ITGA1 and TNFRSF13C, which are bound by HuR, increase surface protein expression compared to their corresponding short 3' UTRs.We propose that during translation the scaffold function of 3' UTRs facilitates binding of proteins to nascent proteins to direct their transport or function--and this role of 3' UTRs can be regulated by ApA.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, 1275 York Ave, New York, New York 10065, USA.

ABSTRACT
About half of human genes use alternative cleavage and polyadenylation (ApA) to generate messenger RNA transcripts that differ in the length of their 3' untranslated regions (3' UTRs) while producing the same protein. Here we show in human cell lines that alternative 3' UTRs differentially regulate the localization of membrane proteins. The long 3' UTR of CD47 enables efficient cell surface expression of CD47 protein, whereas the short 3' UTR primarily localizes CD47 protein to the endoplasmic reticulum. CD47 protein localization occurs post-translationally and independently of RNA localization. In our model of 3' UTR-dependent protein localization, the long 3' UTR of CD47 acts as a scaffold to recruit a protein complex containing the RNA-binding protein HuR (also known as ELAVL1) and SET to the site of translation. This facilitates interaction of SET with the newly translated cytoplasmic domains of CD47 and results in subsequent translocation of CD47 to the plasma membrane via activated RAC1 (ref. 5). We also show that CD47 protein has different functions depending on whether it was generated by the short or long 3' UTR isoforms. Thus, ApA contributes to the functional diversity of the proteome without changing the amino acid sequence. 3' UTR-dependent protein localization has the potential to be a widespread trafficking mechanism for membrane proteins because HuR binds to thousands of mRNAs, and we show that the long 3' UTRs of CD44, ITGA1 and TNFRSF13C, which are bound by HuR, increase surface protein expression compared to their corresponding short 3' UTRs. We propose that during translation the scaffold function of 3' UTRs facilitates binding of proteins to nascent proteins to direct their transport or function--and this role of 3' UTRs can be regulated by ApA.

Show MeSH
Related in: MedlinePlus