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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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ZBP1 plays a role in β-actin mRNA localization. CEFs were transfected with deletion mutants of ZBP1 fused to GFP. Constructs are schematically represented to the left of the table as follows: RRM domains are yellow with green hatching, KH domains are in black, putative nuclear export signal are in green, putative nuclear localization signals are in red, putative MAP kinase site are in yellow. After fixation and permeabilization, in situ hybridization was performed to detect β-actin mRNA. Bars show the percentage of transfected cells counted with β-actin mRNA localized to leading edge. At least 100 cells were counted blind per coverslip in three experiments each. Routinely, 40–50% of nontransfected CEFs show β-actin mRNA localization (unpublished data). (a, 1–179; b, 1–289; c, 1–397; d, 74–576; e, 84–576; f, 189–576; g, 317–576; h, 195–403; i, 195–522; j, 1–576 [ZBP1]).
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fig7: ZBP1 plays a role in β-actin mRNA localization. CEFs were transfected with deletion mutants of ZBP1 fused to GFP. Constructs are schematically represented to the left of the table as follows: RRM domains are yellow with green hatching, KH domains are in black, putative nuclear export signal are in green, putative nuclear localization signals are in red, putative MAP kinase site are in yellow. After fixation and permeabilization, in situ hybridization was performed to detect β-actin mRNA. Bars show the percentage of transfected cells counted with β-actin mRNA localized to leading edge. At least 100 cells were counted blind per coverslip in three experiments each. Routinely, 40–50% of nontransfected CEFs show β-actin mRNA localization (unpublished data). (a, 1–179; b, 1–289; c, 1–397; d, 74–576; e, 84–576; f, 189–576; g, 317–576; h, 195–403; i, 195–522; j, 1–576 [ZBP1]).

Mentions: β-Actin mRNA was localized in 40–50% of nontransfected cells and cells transfected with GFP alone (unpublished data). Overexpression of GFP–ZBP1 had no effect on β-actin localization (Fig. 7). Three of the overexpressed constructs decreased β-actin localization by ∼50%: ΔZBP1 (1–289), ΔZBP1 (189–576), and ΔZBP1 (317–576) (Fig. 7). ΔZBP1 (1–289) contains the two RRM domains and the first KH domain. The other two constructs, ΔZBP1 (189–576) and ΔZBP1 (317–576), contain all four KH domains and the two COOH-terminal KH domains, respectively. There was no obvious difference in the delocalization phenotype elicited by each of the dominant-negative constructs (1–289, 189–576, and 317–576). Each caused β-actin mRNA granules to appear evenly distributed throughout the cytoplasm. Overexpression of the remaining constructs had no effect on β-actin localization in CEFs.


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)

ZBP1 plays a role in β-actin mRNA localization. CEFs were transfected with deletion mutants of ZBP1 fused to GFP. Constructs are schematically represented to the left of the table as follows: RRM domains are yellow with green hatching, KH domains are in black, putative nuclear export signal are in green, putative nuclear localization signals are in red, putative MAP kinase site are in yellow. After fixation and permeabilization, in situ hybridization was performed to detect β-actin mRNA. Bars show the percentage of transfected cells counted with β-actin mRNA localized to leading edge. At least 100 cells were counted blind per coverslip in three experiments each. Routinely, 40–50% of nontransfected CEFs show β-actin mRNA localization (unpublished data). (a, 1–179; b, 1–289; c, 1–397; d, 74–576; e, 84–576; f, 189–576; g, 317–576; h, 195–403; i, 195–522; j, 1–576 [ZBP1]).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172732&req=5

fig7: ZBP1 plays a role in β-actin mRNA localization. CEFs were transfected with deletion mutants of ZBP1 fused to GFP. Constructs are schematically represented to the left of the table as follows: RRM domains are yellow with green hatching, KH domains are in black, putative nuclear export signal are in green, putative nuclear localization signals are in red, putative MAP kinase site are in yellow. After fixation and permeabilization, in situ hybridization was performed to detect β-actin mRNA. Bars show the percentage of transfected cells counted with β-actin mRNA localized to leading edge. At least 100 cells were counted blind per coverslip in three experiments each. Routinely, 40–50% of nontransfected CEFs show β-actin mRNA localization (unpublished data). (a, 1–179; b, 1–289; c, 1–397; d, 74–576; e, 84–576; f, 189–576; g, 317–576; h, 195–403; i, 195–522; j, 1–576 [ZBP1]).
Mentions: β-Actin mRNA was localized in 40–50% of nontransfected cells and cells transfected with GFP alone (unpublished data). Overexpression of GFP–ZBP1 had no effect on β-actin localization (Fig. 7). Three of the overexpressed constructs decreased β-actin localization by ∼50%: ΔZBP1 (1–289), ΔZBP1 (189–576), and ΔZBP1 (317–576) (Fig. 7). ΔZBP1 (1–289) contains the two RRM domains and the first KH domain. The other two constructs, ΔZBP1 (189–576) and ΔZBP1 (317–576), contain all four KH domains and the two COOH-terminal KH domains, respectively. There was no obvious difference in the delocalization phenotype elicited by each of the dominant-negative constructs (1–289, 189–576, and 317–576). Each caused β-actin mRNA granules to appear evenly distributed throughout the cytoplasm. Overexpression of the remaining constructs had no effect on β-actin localization in CEFs.

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