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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.

Show MeSH
Localization phenotypes of GFP–ZBP1 deletion mutants. Red bars indicate putative nuclear localization signals, green bars indicate putative nuclear export signals, and yellow bars indicate putative map kinase sites.
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fig3: Localization phenotypes of GFP–ZBP1 deletion mutants. Red bars indicate putative nuclear localization signals, green bars indicate putative nuclear export signals, and yellow bars indicate putative map kinase sites.

Mentions: We expressed several fragments of ZBP1 fused to GFP in CEFs to define which regions of the protein mediate granule formation, cytoskeletal interaction, and granule localization. Only constructs containing the two COOH-terminal KH domains formed granules and interacted with the cytoskeleton (Fig. 3 and Fig. 2, F–K and F′–K′). Removal of the RRM domains did not affect either granule formation or cytoskeleton association (Fig. 2, F′–K′) in this assay; expression of the four KH domains (189–576) resulted in characteristic ZBP1 granule formation. This activity did not require all four of the KH domains, since a construct containing only the third and fourth KH domains (317–576) formed granules. However, granules formed by constructs lacking the RRM domains distribute evenly throughout the cytoplasm. Specifically, truncated ZBP1 (ΔZBP1; 74–576) retained the characteristic distribution of ZBP1-containing granules. A construct only 10 amino acids shorter, ΔZBP1 (84–576), formed granules that did not accumulate at the cell periphery.


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)

Localization phenotypes of GFP–ZBP1 deletion mutants. Red bars indicate putative nuclear localization signals, green bars indicate putative nuclear export signals, and yellow bars indicate putative map kinase sites.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172732&req=5

fig3: Localization phenotypes of GFP–ZBP1 deletion mutants. Red bars indicate putative nuclear localization signals, green bars indicate putative nuclear export signals, and yellow bars indicate putative map kinase sites.
Mentions: We expressed several fragments of ZBP1 fused to GFP in CEFs to define which regions of the protein mediate granule formation, cytoskeletal interaction, and granule localization. Only constructs containing the two COOH-terminal KH domains formed granules and interacted with the cytoskeleton (Fig. 3 and Fig. 2, F–K and F′–K′). Removal of the RRM domains did not affect either granule formation or cytoskeleton association (Fig. 2, F′–K′) in this assay; expression of the four KH domains (189–576) resulted in characteristic ZBP1 granule formation. This activity did not require all four of the KH domains, since a construct containing only the third and fourth KH domains (317–576) formed granules. However, granules formed by constructs lacking the RRM domains distribute evenly throughout the cytoplasm. Specifically, truncated ZBP1 (ΔZBP1; 74–576) retained the characteristic distribution of ZBP1-containing granules. A construct only 10 amino acids shorter, ΔZBP1 (84–576), formed granules that did not accumulate at the cell periphery.

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