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Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment.

Farina KL, Huttelmaier S, Musunuru K, Darnell R, Singer RH - J. Cell Biol. (2002)

Bottom Line: When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination.RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA.Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

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The KH domains of ZBP1 specifically bind the zipcode of β-actin mRNA. (A) Electrophoretic mobility shift assay of 32P-labeled zipcode RNA with recombinant GST–ZBP1 fragments. ZBP1 fragments were incubated with labeled zipcode in the presence of E. coli tRNA nonspecific competitor followed by heparin. (Lane 1) ZBP1; (lane 2) RRM1–2 (1–195); (lane 3) KH1-KH4 (195–576); (lane 4) GST; (lane 5) no protein. Note that only the full-length protein and the fragment containing the KH domains (lanes 1 and 3) caused a significant shift of the labeled RNA. No binding was observed for the RRM domains or GST (2 and 4). (B) Interaction between zipcode RNA and ZBP1 was reduced by addition of wild-type zipcode RNA but not by mutated zipcode RNA. (C) Nitrocellulose filter binding assay of recombinant GST–ZBP1 fragment affinity for β-actin mRNA zipcode: ZBP1 (1–576), RRM1-2 (1–195), KH1-KH4 (195–576), KH1-2 (195–308), KH3-KH4 (404–576). Recombinant proteins at various concentrations were incubated with 32P-labeled zipcode RNA. Bound probe was detected by Cerenkov counting after binding of the protein–RNA complex to the filter and intensive washing. All fragments containing at least KH domain 3 and 4 bound the zipcode with a Kd in the nM range. (D) To determine the relative affinities of GST–ZBP1 fragments for full-length β-actin, mRNA binding was analyzed in GST pull-down assays. The fraction of bound 32P-labeled human β-actin RNA retained on equal amounts of immobilized GST–ZBP1 proteins were normalized to binding of full-length ZBP1 and plotted according to the fragments used. All proteins containing at least KH3 and 4 bound the full-length mRNA at apparently equal affinity. (E) Human β-actin mRNA coimmunoprecipitates specifically with Flag-tagged ZBP1. RT-PCR amplification of vinculin or β-actin mRNA extracted from supernatant of nontransfected cells (+) or pellets of FLAG–ZBP1 (Z), FLAG–raver1 (R), or mock (−)-transfected cells after immunoprecipitation with anti-Flag (M2). Human β-actin mRNA was specifically enriched in FLAG–ZBP1 pellets, whereas no enrichment of vinculin mRNA was detected. Precipitation of the FLAG-tagged raver1 (left, bottom) or ZBP1 (right, bottom) was verified by Western blotting of pellet fractions using anti-Flag (M2) antibody. (Lane 1) Total cell extract; (lane 2) supernatant after immunoprecipitation; (lane 3) pelleted fraction.
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fig5: The KH domains of ZBP1 specifically bind the zipcode of β-actin mRNA. (A) Electrophoretic mobility shift assay of 32P-labeled zipcode RNA with recombinant GST–ZBP1 fragments. ZBP1 fragments were incubated with labeled zipcode in the presence of E. coli tRNA nonspecific competitor followed by heparin. (Lane 1) ZBP1; (lane 2) RRM1–2 (1–195); (lane 3) KH1-KH4 (195–576); (lane 4) GST; (lane 5) no protein. Note that only the full-length protein and the fragment containing the KH domains (lanes 1 and 3) caused a significant shift of the labeled RNA. No binding was observed for the RRM domains or GST (2 and 4). (B) Interaction between zipcode RNA and ZBP1 was reduced by addition of wild-type zipcode RNA but not by mutated zipcode RNA. (C) Nitrocellulose filter binding assay of recombinant GST–ZBP1 fragment affinity for β-actin mRNA zipcode: ZBP1 (1–576), RRM1-2 (1–195), KH1-KH4 (195–576), KH1-2 (195–308), KH3-KH4 (404–576). Recombinant proteins at various concentrations were incubated with 32P-labeled zipcode RNA. Bound probe was detected by Cerenkov counting after binding of the protein–RNA complex to the filter and intensive washing. All fragments containing at least KH domain 3 and 4 bound the zipcode with a Kd in the nM range. (D) To determine the relative affinities of GST–ZBP1 fragments for full-length β-actin, mRNA binding was analyzed in GST pull-down assays. The fraction of bound 32P-labeled human β-actin RNA retained on equal amounts of immobilized GST–ZBP1 proteins were normalized to binding of full-length ZBP1 and plotted according to the fragments used. All proteins containing at least KH3 and 4 bound the full-length mRNA at apparently equal affinity. (E) Human β-actin mRNA coimmunoprecipitates specifically with Flag-tagged ZBP1. RT-PCR amplification of vinculin or β-actin mRNA extracted from supernatant of nontransfected cells (+) or pellets of FLAG–ZBP1 (Z), FLAG–raver1 (R), or mock (−)-transfected cells after immunoprecipitation with anti-Flag (M2). Human β-actin mRNA was specifically enriched in FLAG–ZBP1 pellets, whereas no enrichment of vinculin mRNA was detected. Precipitation of the FLAG-tagged raver1 (left, bottom) or ZBP1 (right, bottom) was verified by Western blotting of pellet fractions using anti-Flag (M2) antibody. (Lane 1) Total cell extract; (lane 2) supernatant after immunoprecipitation; (lane 3) pelleted fraction.

Mentions: We examined the ability of various constructs to directly bind the zipcode using both electrophoretic mobility shift assays (Fig. 5, A and B) and filter binding assays (Fig. 5, C and D). We determined that the region containing the two COOH-terminal KH domains of ZBP1, not the RRM domains, binds the β-actin mRNA zipcode. Recombinant full-length ZBP1 and fragments comprising KH1-KH4 (ΔZBP1 [195–576]) or KH3-KH4 (ΔZBP1 [404–576]) specifically bind the zipcode, whereas fragments comprising only the RRM domains (ΔZBP1 [1–195]) or the first and second KH domains (ΔZBP1 [195–308]) remained inactive (Fig. 5, A, C, and D). Binding by the KH domains showed the same resistance to RNase T1 digestion as full-length ZBP1 (unpublished data). ZBP1 interactions with the zipcode are stable in the presence of excess nonspecific competitor mRNA and in the presence of a mutated zipcode sequence (Fig. 5, A–C). However, excess zipcode mRNA is capable of competing for the labeled zipcode mRNA in the mobility shift assay (Fig. 5 B).


Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment.

Farina KL, Huttelmaier S, Musunuru K, Darnell R, Singer RH - J. Cell Biol. (2002)

The KH domains of ZBP1 specifically bind the zipcode of β-actin mRNA. (A) Electrophoretic mobility shift assay of 32P-labeled zipcode RNA with recombinant GST–ZBP1 fragments. ZBP1 fragments were incubated with labeled zipcode in the presence of E. coli tRNA nonspecific competitor followed by heparin. (Lane 1) ZBP1; (lane 2) RRM1–2 (1–195); (lane 3) KH1-KH4 (195–576); (lane 4) GST; (lane 5) no protein. Note that only the full-length protein and the fragment containing the KH domains (lanes 1 and 3) caused a significant shift of the labeled RNA. No binding was observed for the RRM domains or GST (2 and 4). (B) Interaction between zipcode RNA and ZBP1 was reduced by addition of wild-type zipcode RNA but not by mutated zipcode RNA. (C) Nitrocellulose filter binding assay of recombinant GST–ZBP1 fragment affinity for β-actin mRNA zipcode: ZBP1 (1–576), RRM1-2 (1–195), KH1-KH4 (195–576), KH1-2 (195–308), KH3-KH4 (404–576). Recombinant proteins at various concentrations were incubated with 32P-labeled zipcode RNA. Bound probe was detected by Cerenkov counting after binding of the protein–RNA complex to the filter and intensive washing. All fragments containing at least KH domain 3 and 4 bound the zipcode with a Kd in the nM range. (D) To determine the relative affinities of GST–ZBP1 fragments for full-length β-actin, mRNA binding was analyzed in GST pull-down assays. The fraction of bound 32P-labeled human β-actin RNA retained on equal amounts of immobilized GST–ZBP1 proteins were normalized to binding of full-length ZBP1 and plotted according to the fragments used. All proteins containing at least KH3 and 4 bound the full-length mRNA at apparently equal affinity. (E) Human β-actin mRNA coimmunoprecipitates specifically with Flag-tagged ZBP1. RT-PCR amplification of vinculin or β-actin mRNA extracted from supernatant of nontransfected cells (+) or pellets of FLAG–ZBP1 (Z), FLAG–raver1 (R), or mock (−)-transfected cells after immunoprecipitation with anti-Flag (M2). Human β-actin mRNA was specifically enriched in FLAG–ZBP1 pellets, whereas no enrichment of vinculin mRNA was detected. Precipitation of the FLAG-tagged raver1 (left, bottom) or ZBP1 (right, bottom) was verified by Western blotting of pellet fractions using anti-Flag (M2) antibody. (Lane 1) Total cell extract; (lane 2) supernatant after immunoprecipitation; (lane 3) pelleted fraction.
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fig5: The KH domains of ZBP1 specifically bind the zipcode of β-actin mRNA. (A) Electrophoretic mobility shift assay of 32P-labeled zipcode RNA with recombinant GST–ZBP1 fragments. ZBP1 fragments were incubated with labeled zipcode in the presence of E. coli tRNA nonspecific competitor followed by heparin. (Lane 1) ZBP1; (lane 2) RRM1–2 (1–195); (lane 3) KH1-KH4 (195–576); (lane 4) GST; (lane 5) no protein. Note that only the full-length protein and the fragment containing the KH domains (lanes 1 and 3) caused a significant shift of the labeled RNA. No binding was observed for the RRM domains or GST (2 and 4). (B) Interaction between zipcode RNA and ZBP1 was reduced by addition of wild-type zipcode RNA but not by mutated zipcode RNA. (C) Nitrocellulose filter binding assay of recombinant GST–ZBP1 fragment affinity for β-actin mRNA zipcode: ZBP1 (1–576), RRM1-2 (1–195), KH1-KH4 (195–576), KH1-2 (195–308), KH3-KH4 (404–576). Recombinant proteins at various concentrations were incubated with 32P-labeled zipcode RNA. Bound probe was detected by Cerenkov counting after binding of the protein–RNA complex to the filter and intensive washing. All fragments containing at least KH domain 3 and 4 bound the zipcode with a Kd in the nM range. (D) To determine the relative affinities of GST–ZBP1 fragments for full-length β-actin, mRNA binding was analyzed in GST pull-down assays. The fraction of bound 32P-labeled human β-actin RNA retained on equal amounts of immobilized GST–ZBP1 proteins were normalized to binding of full-length ZBP1 and plotted according to the fragments used. All proteins containing at least KH3 and 4 bound the full-length mRNA at apparently equal affinity. (E) Human β-actin mRNA coimmunoprecipitates specifically with Flag-tagged ZBP1. RT-PCR amplification of vinculin or β-actin mRNA extracted from supernatant of nontransfected cells (+) or pellets of FLAG–ZBP1 (Z), FLAG–raver1 (R), or mock (−)-transfected cells after immunoprecipitation with anti-Flag (M2). Human β-actin mRNA was specifically enriched in FLAG–ZBP1 pellets, whereas no enrichment of vinculin mRNA was detected. Precipitation of the FLAG-tagged raver1 (left, bottom) or ZBP1 (right, bottom) was verified by Western blotting of pellet fractions using anti-Flag (M2) antibody. (Lane 1) Total cell extract; (lane 2) supernatant after immunoprecipitation; (lane 3) pelleted fraction.
Mentions: We examined the ability of various constructs to directly bind the zipcode using both electrophoretic mobility shift assays (Fig. 5, A and B) and filter binding assays (Fig. 5, C and D). We determined that the region containing the two COOH-terminal KH domains of ZBP1, not the RRM domains, binds the β-actin mRNA zipcode. Recombinant full-length ZBP1 and fragments comprising KH1-KH4 (ΔZBP1 [195–576]) or KH3-KH4 (ΔZBP1 [404–576]) specifically bind the zipcode, whereas fragments comprising only the RRM domains (ΔZBP1 [1–195]) or the first and second KH domains (ΔZBP1 [195–308]) remained inactive (Fig. 5, A, C, and D). Binding by the KH domains showed the same resistance to RNase T1 digestion as full-length ZBP1 (unpublished data). ZBP1 interactions with the zipcode are stable in the presence of excess nonspecific competitor mRNA and in the presence of a mutated zipcode sequence (Fig. 5, A–C). However, excess zipcode mRNA is capable of competing for the labeled zipcode mRNA in the mobility shift assay (Fig. 5 B).

Bottom Line: When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination.RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA.Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

ABSTRACT
Chicken embryo fibroblasts (CEFs) localize beta-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of beta-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically binds to the zipcode. Both localize to the lamellipodia of polarized CEFs. ZBP1 and its homologues contain two NH2-terminal RNA recognition motifs (RRMs) and four COOH-terminal hnRNP K homology (KH) domains. By using ZBP1 truncations fused to GFP in conjunction with in situ hybridization analysis, we have determined that KH domains three and four were responsible for granule formation and cytoskeletal association. When the NH2 terminus was deleted, granules formed by the KH domains alone did not accumulate at the leading edge, suggesting a role for the NH2 terminus in targeting transport granules to their destination. RNA binding studies were used to show that the third and fourth KH domains, not the RRM domains, bind the zipcode of beta-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized beta-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both beta-actin mRNA localization and cell motility.

Show MeSH