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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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3'UTR-dependent protein localization (UDPL) depends on HuR, SET and RAC1, and mediates surface localization of membrane proteins(A) Model of UDPL. HuR binds to the long 3'UTR and recruits SET. During translation of CD47 mRNA this protein complex is targeted to the endoplasmic reticulum (ER) surface where SET binds to the newly translated cytoplasmic domains of CD47. This step likely requires energy. SET interacts with RAC1 and active RAC1 translocates SET and CD47 to the plasma membrane.(B) FACS analysis of endogenous CD47 protein expression in HEK293 cells. Left panel is after transfection of control shRNA (shCo) or shRNAs against HuR (shown are all GFP+ cells). Middle and right panels depict cells stably expressing the indicated shRNAs. Surface CD47 (top) and total CD47 protein (bottom) were measured.(C) 3'-seq analysis for CD44 in HEK293 cells and for TNFRSF13C in B-LCL cells, as shown in Fig. 1b. FACS analysis of endogenous CD44 protein in HEK293 cells (left) and endogenous BAFFR protein in SHSY-5Y cells (right) shown as in b (left).(D) Left, FACS analysis of GFP after transfection of constructs containing a signal peptide and GFP fused to the TMD and C terminus of CD44 and either the long 39UTR (dark blue line) or the short 3'UTR(light blue line) in U251 cells. Right, as in left panel, but for BAFFR with transfection into HeLa cells.(E) FACS analysis of GFP expression shows that HuR-BS is sufficient for surface localization of GFP-TM (dark blue line). Deletion of the binding sites from the HuR-BS construct (HuR-BSΔ) abrogates GFP surface expression (light blue line).For b, c, d and e surface and total protein expression were determined and shown as in Fig. 1g. Representative images from three (sh2 HuR, n = 5) biological replicates.
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Figure 2: 3'UTR-dependent protein localization (UDPL) depends on HuR, SET and RAC1, and mediates surface localization of membrane proteins(A) Model of UDPL. HuR binds to the long 3'UTR and recruits SET. During translation of CD47 mRNA this protein complex is targeted to the endoplasmic reticulum (ER) surface where SET binds to the newly translated cytoplasmic domains of CD47. This step likely requires energy. SET interacts with RAC1 and active RAC1 translocates SET and CD47 to the plasma membrane.(B) FACS analysis of endogenous CD47 protein expression in HEK293 cells. Left panel is after transfection of control shRNA (shCo) or shRNAs against HuR (shown are all GFP+ cells). Middle and right panels depict cells stably expressing the indicated shRNAs. Surface CD47 (top) and total CD47 protein (bottom) were measured.(C) 3'-seq analysis for CD44 in HEK293 cells and for TNFRSF13C in B-LCL cells, as shown in Fig. 1b. FACS analysis of endogenous CD44 protein in HEK293 cells (left) and endogenous BAFFR protein in SHSY-5Y cells (right) shown as in b (left).(D) Left, FACS analysis of GFP after transfection of constructs containing a signal peptide and GFP fused to the TMD and C terminus of CD44 and either the long 39UTR (dark blue line) or the short 3'UTR(light blue line) in U251 cells. Right, as in left panel, but for BAFFR with transfection into HeLa cells.(E) FACS analysis of GFP expression shows that HuR-BS is sufficient for surface localization of GFP-TM (dark blue line). Deletion of the binding sites from the HuR-BS construct (HuR-BSΔ) abrogates GFP surface expression (light blue line).For b, c, d and e surface and total protein expression were determined and shown as in Fig. 1g. Representative images from three (sh2 HuR, n = 5) biological replicates.

Mentions: To address the mechanism of 3'UTR-dependent protein localization (UDPL; Fig. 2a), we reasoned there must be an RNA-binding protein (RBP) that binds to the long, but not the short, 3'UTR of CD47. The long 3'UTR of CD47 contains many uridine-rich elements (see later) which are potentially bound by HuR 6–9. HuR is known for its role in mRNA stabilization and translation activation 7,13,14. However, HuR KD by shRNAs did not affect CD47 mRNA abundance or isoform levels, nor did it affect total CD47 protein levels (Fig. 2b, bottom and Extended Data Fig. 1d, 2a–c). But, strikingly, KD of HuR reduced CD47 surface expression (Fig. 2b, top and Extended Data Fig. 2c). This suggests that for CD47, HuR mediates protein localization post-translationally.


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

Berkovits BD, Mayr C - Nature (2015)

3'UTR-dependent protein localization (UDPL) depends on HuR, SET and RAC1, and mediates surface localization of membrane proteins(A) Model of UDPL. HuR binds to the long 3'UTR and recruits SET. During translation of CD47 mRNA this protein complex is targeted to the endoplasmic reticulum (ER) surface where SET binds to the newly translated cytoplasmic domains of CD47. This step likely requires energy. SET interacts with RAC1 and active RAC1 translocates SET and CD47 to the plasma membrane.(B) FACS analysis of endogenous CD47 protein expression in HEK293 cells. Left panel is after transfection of control shRNA (shCo) or shRNAs against HuR (shown are all GFP+ cells). Middle and right panels depict cells stably expressing the indicated shRNAs. Surface CD47 (top) and total CD47 protein (bottom) were measured.(C) 3'-seq analysis for CD44 in HEK293 cells and for TNFRSF13C in B-LCL cells, as shown in Fig. 1b. FACS analysis of endogenous CD44 protein in HEK293 cells (left) and endogenous BAFFR protein in SHSY-5Y cells (right) shown as in b (left).(D) Left, FACS analysis of GFP after transfection of constructs containing a signal peptide and GFP fused to the TMD and C terminus of CD44 and either the long 39UTR (dark blue line) or the short 3'UTR(light blue line) in U251 cells. Right, as in left panel, but for BAFFR with transfection into HeLa cells.(E) FACS analysis of GFP expression shows that HuR-BS is sufficient for surface localization of GFP-TM (dark blue line). Deletion of the binding sites from the HuR-BS construct (HuR-BSΔ) abrogates GFP surface expression (light blue line).For b, c, d and e surface and total protein expression were determined and shown as in Fig. 1g. Representative images from three (sh2 HuR, n = 5) biological replicates.
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Figure 2: 3'UTR-dependent protein localization (UDPL) depends on HuR, SET and RAC1, and mediates surface localization of membrane proteins(A) Model of UDPL. HuR binds to the long 3'UTR and recruits SET. During translation of CD47 mRNA this protein complex is targeted to the endoplasmic reticulum (ER) surface where SET binds to the newly translated cytoplasmic domains of CD47. This step likely requires energy. SET interacts with RAC1 and active RAC1 translocates SET and CD47 to the plasma membrane.(B) FACS analysis of endogenous CD47 protein expression in HEK293 cells. Left panel is after transfection of control shRNA (shCo) or shRNAs against HuR (shown are all GFP+ cells). Middle and right panels depict cells stably expressing the indicated shRNAs. Surface CD47 (top) and total CD47 protein (bottom) were measured.(C) 3'-seq analysis for CD44 in HEK293 cells and for TNFRSF13C in B-LCL cells, as shown in Fig. 1b. FACS analysis of endogenous CD44 protein in HEK293 cells (left) and endogenous BAFFR protein in SHSY-5Y cells (right) shown as in b (left).(D) Left, FACS analysis of GFP after transfection of constructs containing a signal peptide and GFP fused to the TMD and C terminus of CD44 and either the long 39UTR (dark blue line) or the short 3'UTR(light blue line) in U251 cells. Right, as in left panel, but for BAFFR with transfection into HeLa cells.(E) FACS analysis of GFP expression shows that HuR-BS is sufficient for surface localization of GFP-TM (dark blue line). Deletion of the binding sites from the HuR-BS construct (HuR-BSΔ) abrogates GFP surface expression (light blue line).For b, c, d and e surface and total protein expression were determined and shown as in Fig. 1g. Representative images from three (sh2 HuR, n = 5) biological replicates.
Mentions: To address the mechanism of 3'UTR-dependent protein localization (UDPL; Fig. 2a), we reasoned there must be an RNA-binding protein (RBP) that binds to the long, but not the short, 3'UTR of CD47. The long 3'UTR of CD47 contains many uridine-rich elements (see later) which are potentially bound by HuR 6–9. HuR is known for its role in mRNA stabilization and translation activation 7,13,14. However, HuR KD by shRNAs did not affect CD47 mRNA abundance or isoform levels, nor did it affect total CD47 protein levels (Fig. 2b, bottom and Extended Data Fig. 1d, 2a–c). But, strikingly, KD of HuR reduced CD47 surface expression (Fig. 2b, top and Extended Data Fig. 2c). This suggests that for CD47, HuR mediates protein localization post-translationally.

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