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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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Selection of ZBP1 (KH3-KH4) ligands using RNA selection amplification (SELEX). (A) Lowercase letters are fixed sequences present in RNA library clones. Boxed sequences indicate consensus sequence. (B) Interaction of ZBP1 with zipcode RNA was reduced by addition of 20-mer oligonucleotides that bear ACACCC repeats of the zipcode or consensus SELEX sequences (S1).
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fig6: Selection of ZBP1 (KH3-KH4) ligands using RNA selection amplification (SELEX). (A) Lowercase letters are fixed sequences present in RNA library clones. Boxed sequences indicate consensus sequence. (B) Interaction of ZBP1 with zipcode RNA was reduced by addition of 20-mer oligonucleotides that bear ACACCC repeats of the zipcode or consensus SELEX sequences (S1).

Mentions: We used RNA selection amplification (SELEX) (Ellington and Szostak, 1990; Tuerk and Gold, 1990) to determine the sequence specificity of the ZBP1 zipcode RNA binding region. For this experiment, we used the deletion construct ΔZBP1 (404–576) in eight rounds of selection amplification. This construct comprises the third and fourth KH domains and the remaining COOH-terminal amino acids of the protein. We chose this construct because it suffices for high affinity zipcode binding in filter binding assays. After eight rounds of selection, the resultant RNA pool was cloned and sequenced. 71% percent of the clones (12 out of 17) could be organized into a strong consensus group (Fig. 6 A). These clones contained the 11-mer 5′-AAGCACCCGTT-3′ or some close variant of this sequence. In addition to the clones that are included in Fig. 6 A, four clones harbored the sequence 5′-CCGCACG-3′ (unpublished data). One clone was quite random and harbored no enrichments. The zipcode of β-actin mRNA, a putative stem-loop, contains repeats of an almost identical element in its predicted loop region, (5′-ACACCC-3′)2 (Kislauskis et al., 1993; Ross et al., 1997). Thus, the ZBP1 KH3-KH4 domains appear to exhibit a preference for the sequence 5′-RCACCC-3′ (R denotes pyrimidine) both in selected ligands and in a physiological ligand. To compare the relative affinities of the ACACCC repeats of the zipcode versus the SELEX consensus sequence for ZBP1, competition experiments were performed with 20-mer oligoribonucleotides containing portions of the zipcode or the SELEX consensus sequence (Fig. 6 B, S1). The SELEX consensus was equally as efficient at competing for binding with ZBP1 as were the zipcode sequences containing ACACCC (Fig. 6 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)

Selection of ZBP1 (KH3-KH4) ligands using RNA selection amplification (SELEX). (A) Lowercase letters are fixed sequences present in RNA library clones. Boxed sequences indicate consensus sequence. (B) Interaction of ZBP1 with zipcode RNA was reduced by addition of 20-mer oligonucleotides that bear ACACCC repeats of the zipcode or consensus SELEX sequences (S1).
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Related In: Results  -  Collection

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fig6: Selection of ZBP1 (KH3-KH4) ligands using RNA selection amplification (SELEX). (A) Lowercase letters are fixed sequences present in RNA library clones. Boxed sequences indicate consensus sequence. (B) Interaction of ZBP1 with zipcode RNA was reduced by addition of 20-mer oligonucleotides that bear ACACCC repeats of the zipcode or consensus SELEX sequences (S1).
Mentions: We used RNA selection amplification (SELEX) (Ellington and Szostak, 1990; Tuerk and Gold, 1990) to determine the sequence specificity of the ZBP1 zipcode RNA binding region. For this experiment, we used the deletion construct ΔZBP1 (404–576) in eight rounds of selection amplification. This construct comprises the third and fourth KH domains and the remaining COOH-terminal amino acids of the protein. We chose this construct because it suffices for high affinity zipcode binding in filter binding assays. After eight rounds of selection, the resultant RNA pool was cloned and sequenced. 71% percent of the clones (12 out of 17) could be organized into a strong consensus group (Fig. 6 A). These clones contained the 11-mer 5′-AAGCACCCGTT-3′ or some close variant of this sequence. In addition to the clones that are included in Fig. 6 A, four clones harbored the sequence 5′-CCGCACG-3′ (unpublished data). One clone was quite random and harbored no enrichments. The zipcode of β-actin mRNA, a putative stem-loop, contains repeats of an almost identical element in its predicted loop region, (5′-ACACCC-3′)2 (Kislauskis et al., 1993; Ross et al., 1997). Thus, the ZBP1 KH3-KH4 domains appear to exhibit a preference for the sequence 5′-RCACCC-3′ (R denotes pyrimidine) both in selected ligands and in a physiological ligand. To compare the relative affinities of the ACACCC repeats of the zipcode versus the SELEX consensus sequence for ZBP1, competition experiments were performed with 20-mer oligoribonucleotides containing portions of the zipcode or the SELEX consensus sequence (Fig. 6 B, S1). The SELEX consensus was equally as efficient at competing for binding with ZBP1 as were the zipcode sequences containing ACACCC (Fig. 6 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