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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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Local recruitment of SET to the site of translation is required for UDPL(A) Western blot of cells used in Fig. 3b shows the amount of overexpression achieved by transfection of MS2-mC-SET or MS2-mC-HuR [constructs, see (b)]. Left, anti-SET detects endogenous expression of SET as well as overexpressed SET. Right, anti-HuR detects endogenous HuR and overexpressed HuR. Actin was used as loading control. Anti-HuR and anti-SET were used on the same blot. Actin as loading control was performed once. The marker is shown in kD. Asterisk indicates unspecific band. mC, mCherry.(B) The top construct depicts GFP-TM-SU (Fig. 1e) and the bottom construct shows a fusion of MS2 coat protein (MS2), mC (red) and HuR or SET, respectively. Overexpression of HuR or SET compared with expression of MS2-mC alone does not change surface or total GFP expression, when co-transfected with GFP-TM-SU (without the addition of MS2-binding sites to the SU isoform) as shown by FACS analysis. Surface expression (top) and total expression (bottom) in HEK293 cells are shown. Values for MFI are shown in parentheses. Unstained cells are shown in grey. Representative histograms from n = 2 experiments are shown.(C) FACS analysis of cells used in Fig. 3b. MS2-binding sites (MS2-BS, RNA stem loops) were added to GFP-TM-SU (and the proximal polyadenylation signal was mutated) to obtain GFP-TM-SU-MS2-BS. Transfection of MS2-mC-HuR (left, dark purple line) or MS2-mC-SET (right, dark purple line) increases surface GFP expression compared with transfection of MS2-mC (light purple line), when GFP-TM-SU-MS2-BS is co-transfected. Thus, tethering of HuR or SET to the short 3'UTR of GFP-TM localizes GFP to the cell surface without changing total GFP expression. Histograms are shown as in (b). Representative histograms from n = 5 experiments are shown.(D) As in (c), but tethering was impaired by omission of the MS2 coat protein. Histograms are shown as in (b). Representative histograms from n = 2 experiments are shown.Summary of the tethering experiment: To tether SET or HuR to the 3'UTR (which brings it close to the site of translation through the scaffold function of the 3'UTR), we added MS2-binding sites (MS2-BS) to GFP-TM-SU (c). MS2-binding sites are derived from the bacteriophage MS2 and form RNA stem loops. The capsid protein of MS2 (here, called MS2) specifically recognizes these MS2 stem loops. Constructs were generated containing MS2 fused to mC and then either HuR, SET or with no further coding sequence (Fig. 3c). Co-expression of these constructs with the construct containing the short UTR and MS2-binding sites results in recruitment of SET or HuR to the short 3'UTR of GFP-TM. The cells that express MS2 fused to only mC localize GFP to the endoplasmic reticulum, but constructs containing MS2 fusions to HuR or SET localize GFP primarily to the cell surface (Fig. 3b and Extended Data Fig. 5c). Omitting either the MS2 or MS2-binding sites from the experiment abrogates surface localization (Extended Data Fig. 5b, d).
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Figure 9: Local recruitment of SET to the site of translation is required for UDPL(A) Western blot of cells used in Fig. 3b shows the amount of overexpression achieved by transfection of MS2-mC-SET or MS2-mC-HuR [constructs, see (b)]. Left, anti-SET detects endogenous expression of SET as well as overexpressed SET. Right, anti-HuR detects endogenous HuR and overexpressed HuR. Actin was used as loading control. Anti-HuR and anti-SET were used on the same blot. Actin as loading control was performed once. The marker is shown in kD. Asterisk indicates unspecific band. mC, mCherry.(B) The top construct depicts GFP-TM-SU (Fig. 1e) and the bottom construct shows a fusion of MS2 coat protein (MS2), mC (red) and HuR or SET, respectively. Overexpression of HuR or SET compared with expression of MS2-mC alone does not change surface or total GFP expression, when co-transfected with GFP-TM-SU (without the addition of MS2-binding sites to the SU isoform) as shown by FACS analysis. Surface expression (top) and total expression (bottom) in HEK293 cells are shown. Values for MFI are shown in parentheses. Unstained cells are shown in grey. Representative histograms from n = 2 experiments are shown.(C) FACS analysis of cells used in Fig. 3b. MS2-binding sites (MS2-BS, RNA stem loops) were added to GFP-TM-SU (and the proximal polyadenylation signal was mutated) to obtain GFP-TM-SU-MS2-BS. Transfection of MS2-mC-HuR (left, dark purple line) or MS2-mC-SET (right, dark purple line) increases surface GFP expression compared with transfection of MS2-mC (light purple line), when GFP-TM-SU-MS2-BS is co-transfected. Thus, tethering of HuR or SET to the short 3'UTR of GFP-TM localizes GFP to the cell surface without changing total GFP expression. Histograms are shown as in (b). Representative histograms from n = 5 experiments are shown.(D) As in (c), but tethering was impaired by omission of the MS2 coat protein. Histograms are shown as in (b). Representative histograms from n = 2 experiments are shown.Summary of the tethering experiment: To tether SET or HuR to the 3'UTR (which brings it close to the site of translation through the scaffold function of the 3'UTR), we added MS2-binding sites (MS2-BS) to GFP-TM-SU (c). MS2-binding sites are derived from the bacteriophage MS2 and form RNA stem loops. The capsid protein of MS2 (here, called MS2) specifically recognizes these MS2 stem loops. Constructs were generated containing MS2 fused to mC and then either HuR, SET or with no further coding sequence (Fig. 3c). Co-expression of these constructs with the construct containing the short UTR and MS2-binding sites results in recruitment of SET or HuR to the short 3'UTR of GFP-TM. The cells that express MS2 fused to only mC localize GFP to the endoplasmic reticulum, but constructs containing MS2 fusions to HuR or SET localize GFP primarily to the cell surface (Fig. 3b and Extended Data Fig. 5c). Omitting either the MS2 or MS2-binding sites from the experiment abrogates surface localization (Extended Data Fig. 5b, d).

Mentions: Next, we investigated each step of our UDPL model in more detail. We demonstrated by RNA immunoprecipitation (RNA-IP) that HuR binds to the HuR-BS and to the LU but not to the SU isoform of CD47 (Fig. 3a, left). SET also associates with the long 3'UTR of CD47, which is dependent on HuR (Fig. 3a, right). SET or HuR overexpression was insufficient to localize GFP-TM-SU to the cell surface (Extended Data Fig. 5a, b). However, tethering of SET or HuR to the short 3'UTR isoform of CD47 was sufficient to redirect GFP-TM localization from the ER to the plasma membrane (Fig. 3b and Extended Data Fig. 5c, d; see Extended Data Fig. 5 for experimental details). This indicates that local recruitment of SET to the site of translation, mediated by the scaffold function of the long 3'UTR, is required for UDPL.


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

Berkovits BD, Mayr C - Nature (2015)

Local recruitment of SET to the site of translation is required for UDPL(A) Western blot of cells used in Fig. 3b shows the amount of overexpression achieved by transfection of MS2-mC-SET or MS2-mC-HuR [constructs, see (b)]. Left, anti-SET detects endogenous expression of SET as well as overexpressed SET. Right, anti-HuR detects endogenous HuR and overexpressed HuR. Actin was used as loading control. Anti-HuR and anti-SET were used on the same blot. Actin as loading control was performed once. The marker is shown in kD. Asterisk indicates unspecific band. mC, mCherry.(B) The top construct depicts GFP-TM-SU (Fig. 1e) and the bottom construct shows a fusion of MS2 coat protein (MS2), mC (red) and HuR or SET, respectively. Overexpression of HuR or SET compared with expression of MS2-mC alone does not change surface or total GFP expression, when co-transfected with GFP-TM-SU (without the addition of MS2-binding sites to the SU isoform) as shown by FACS analysis. Surface expression (top) and total expression (bottom) in HEK293 cells are shown. Values for MFI are shown in parentheses. Unstained cells are shown in grey. Representative histograms from n = 2 experiments are shown.(C) FACS analysis of cells used in Fig. 3b. MS2-binding sites (MS2-BS, RNA stem loops) were added to GFP-TM-SU (and the proximal polyadenylation signal was mutated) to obtain GFP-TM-SU-MS2-BS. Transfection of MS2-mC-HuR (left, dark purple line) or MS2-mC-SET (right, dark purple line) increases surface GFP expression compared with transfection of MS2-mC (light purple line), when GFP-TM-SU-MS2-BS is co-transfected. Thus, tethering of HuR or SET to the short 3'UTR of GFP-TM localizes GFP to the cell surface without changing total GFP expression. Histograms are shown as in (b). Representative histograms from n = 5 experiments are shown.(D) As in (c), but tethering was impaired by omission of the MS2 coat protein. Histograms are shown as in (b). Representative histograms from n = 2 experiments are shown.Summary of the tethering experiment: To tether SET or HuR to the 3'UTR (which brings it close to the site of translation through the scaffold function of the 3'UTR), we added MS2-binding sites (MS2-BS) to GFP-TM-SU (c). MS2-binding sites are derived from the bacteriophage MS2 and form RNA stem loops. The capsid protein of MS2 (here, called MS2) specifically recognizes these MS2 stem loops. Constructs were generated containing MS2 fused to mC and then either HuR, SET or with no further coding sequence (Fig. 3c). Co-expression of these constructs with the construct containing the short UTR and MS2-binding sites results in recruitment of SET or HuR to the short 3'UTR of GFP-TM. The cells that express MS2 fused to only mC localize GFP to the endoplasmic reticulum, but constructs containing MS2 fusions to HuR or SET localize GFP primarily to the cell surface (Fig. 3b and Extended Data Fig. 5c). Omitting either the MS2 or MS2-binding sites from the experiment abrogates surface localization (Extended Data Fig. 5b, d).
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Figure 9: Local recruitment of SET to the site of translation is required for UDPL(A) Western blot of cells used in Fig. 3b shows the amount of overexpression achieved by transfection of MS2-mC-SET or MS2-mC-HuR [constructs, see (b)]. Left, anti-SET detects endogenous expression of SET as well as overexpressed SET. Right, anti-HuR detects endogenous HuR and overexpressed HuR. Actin was used as loading control. Anti-HuR and anti-SET were used on the same blot. Actin as loading control was performed once. The marker is shown in kD. Asterisk indicates unspecific band. mC, mCherry.(B) The top construct depicts GFP-TM-SU (Fig. 1e) and the bottom construct shows a fusion of MS2 coat protein (MS2), mC (red) and HuR or SET, respectively. Overexpression of HuR or SET compared with expression of MS2-mC alone does not change surface or total GFP expression, when co-transfected with GFP-TM-SU (without the addition of MS2-binding sites to the SU isoform) as shown by FACS analysis. Surface expression (top) and total expression (bottom) in HEK293 cells are shown. Values for MFI are shown in parentheses. Unstained cells are shown in grey. Representative histograms from n = 2 experiments are shown.(C) FACS analysis of cells used in Fig. 3b. MS2-binding sites (MS2-BS, RNA stem loops) were added to GFP-TM-SU (and the proximal polyadenylation signal was mutated) to obtain GFP-TM-SU-MS2-BS. Transfection of MS2-mC-HuR (left, dark purple line) or MS2-mC-SET (right, dark purple line) increases surface GFP expression compared with transfection of MS2-mC (light purple line), when GFP-TM-SU-MS2-BS is co-transfected. Thus, tethering of HuR or SET to the short 3'UTR of GFP-TM localizes GFP to the cell surface without changing total GFP expression. Histograms are shown as in (b). Representative histograms from n = 5 experiments are shown.(D) As in (c), but tethering was impaired by omission of the MS2 coat protein. Histograms are shown as in (b). Representative histograms from n = 2 experiments are shown.Summary of the tethering experiment: To tether SET or HuR to the 3'UTR (which brings it close to the site of translation through the scaffold function of the 3'UTR), we added MS2-binding sites (MS2-BS) to GFP-TM-SU (c). MS2-binding sites are derived from the bacteriophage MS2 and form RNA stem loops. The capsid protein of MS2 (here, called MS2) specifically recognizes these MS2 stem loops. Constructs were generated containing MS2 fused to mC and then either HuR, SET or with no further coding sequence (Fig. 3c). Co-expression of these constructs with the construct containing the short UTR and MS2-binding sites results in recruitment of SET or HuR to the short 3'UTR of GFP-TM. The cells that express MS2 fused to only mC localize GFP to the endoplasmic reticulum, but constructs containing MS2 fusions to HuR or SET localize GFP primarily to the cell surface (Fig. 3b and Extended Data Fig. 5c). Omitting either the MS2 or MS2-binding sites from the experiment abrogates surface localization (Extended Data Fig. 5b, d).
Mentions: Next, we investigated each step of our UDPL model in more detail. We demonstrated by RNA immunoprecipitation (RNA-IP) that HuR binds to the HuR-BS and to the LU but not to the SU isoform of CD47 (Fig. 3a, left). SET also associates with the long 3'UTR of CD47, which is dependent on HuR (Fig. 3a, right). SET or HuR overexpression was insufficient to localize GFP-TM-SU to the cell surface (Extended Data Fig. 5a, b). However, tethering of SET or HuR to the short 3'UTR isoform of CD47 was sufficient to redirect GFP-TM localization from the ER to the plasma membrane (Fig. 3b and Extended Data Fig. 5c, d; see Extended Data Fig. 5 for experimental details). This indicates that local recruitment of SET to the site of translation, mediated by the scaffold function of the long 3'UTR, is required for UDPL.

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