Two ZBP1 KH Domains Facilitate β-Actin mRNA Localization, Granule Formation, and Cytoskeletal Attachment
Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-actin mRNA requires a cis localization element (zipcode) and involves zipcode binding protein 1 (ZBP1), a protein that specifically...
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| Veröffentlicht in: | The Journal of cell biology 2003-01, Vol.160 (1), p.77-87 |
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| creator | Farina, Kim L. Hüttelmaier, Stefan Musunuru, Kiran Darnell, Robert Singer, Robert H. |
| description | Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-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 β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility. |
| doi_str_mv | 10.1083/jcb.200206003 |
| format | Article |
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The localization of β-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 β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility.</description><identifier>ISSN: 0021-9525</identifier><identifier>EISSN: 1540-8140</identifier><identifier>DOI: 10.1083/jcb.200206003</identifier><identifier>PMID: 12507992</identifier><identifier>CODEN: JCLBA3</identifier><language>eng</language><publisher>United States: Rockefeller University Press</publisher><subject>Actins ; Actins - genetics ; Actins - metabolism ; Amino Acid Sequence ; Animals ; Avian Proteins ; Cell motility ; Cell Movement ; Cells ; Chick Embryo ; Cytoskeleton ; Cytoskeleton - metabolism ; Dose-Response Relationship, Drug ; Fibroblasts ; Gene Deletion ; Glutathione Transferase - metabolism ; In Situ Hybridization ; In Situ Hybridization, Fluorescence ; Messenger RNA ; Microfilaments ; Microscopy, Fluorescence ; Molecular Sequence Data ; Neurons ; Neurotransmitters ; Perceptual localization ; Phenotype ; Precipitin Tests ; Protein Structure, Tertiary ; Recombinant Proteins - metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Ribonucleic acid ; RNA ; RNA - metabolism ; RNA, Messenger - metabolism ; RNA-Binding Proteins - chemistry ; RNA-Binding Proteins - metabolism ; Sequence Homology, Amino Acid ; Skeletal system ; Transcription, Genetic ; Transfection</subject><ispartof>The Journal of cell biology, 2003-01, Vol.160 (1), p.77-87</ispartof><rights>Copyright 2003 The Rockefeller University Press</rights><rights>Copyright Rockefeller University Press Jan 6, 2003</rights><rights>Copyright © 2003, The Rockefeller University Press</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c431t-d12fc2e2dbf4cd27337ab53e8d0599f806e2f13da225fd9e338ecf72de29f2113</citedby><cites>FETCH-LOGICAL-c431t-d12fc2e2dbf4cd27337ab53e8d0599f806e2f13da225fd9e338ecf72de29f2113</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,776,780,881,27901,27902</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12507992$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Farina, Kim L.</creatorcontrib><creatorcontrib>Hüttelmaier, Stefan</creatorcontrib><creatorcontrib>Musunuru, Kiran</creatorcontrib><creatorcontrib>Darnell, Robert</creatorcontrib><creatorcontrib>Singer, Robert H.</creatorcontrib><title>Two ZBP1 KH Domains Facilitate β-Actin mRNA Localization, Granule Formation, and Cytoskeletal Attachment</title><title>The Journal of cell biology</title><addtitle>J Cell Biol</addtitle><description>Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-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 β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility.</description><subject>Actins</subject><subject>Actins - genetics</subject><subject>Actins - metabolism</subject><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Avian Proteins</subject><subject>Cell motility</subject><subject>Cell Movement</subject><subject>Cells</subject><subject>Chick Embryo</subject><subject>Cytoskeleton</subject><subject>Cytoskeleton - metabolism</subject><subject>Dose-Response Relationship, Drug</subject><subject>Fibroblasts</subject><subject>Gene Deletion</subject><subject>Glutathione Transferase - metabolism</subject><subject>In Situ Hybridization</subject><subject>In Situ Hybridization, Fluorescence</subject><subject>Messenger RNA</subject><subject>Microfilaments</subject><subject>Microscopy, Fluorescence</subject><subject>Molecular Sequence Data</subject><subject>Neurons</subject><subject>Neurotransmitters</subject><subject>Perceptual localization</subject><subject>Phenotype</subject><subject>Precipitin Tests</subject><subject>Protein Structure, Tertiary</subject><subject>Recombinant Proteins - metabolism</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA - metabolism</subject><subject>RNA, Messenger - metabolism</subject><subject>RNA-Binding Proteins - chemistry</subject><subject>RNA-Binding Proteins - metabolism</subject><subject>Sequence Homology, Amino Acid</subject><subject>Skeletal system</subject><subject>Transcription, Genetic</subject><subject>Transfection</subject><issn>0021-9525</issn><issn>1540-8140</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2003</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkc1uEzEUhS0EoqGwZIeQxYIVU-zrmcx4gxQCaRERIFQ2bCzHP9TBYxfbAyqPxYPwTDhK1AKrK5376dxzdRB6SMkJJQN7vlWbEyAEyJwQdgvNaNeSZqAtuY1mVaYN76A7Qvdy3hJC2r5ld9ERhY70nMMMufMfEX9--YHit2f4VRylCxmvpHLeFVkM_v2rWajiAh4_vlvgdVTSu5-yuBie4dMkw-QNXsU0HiQZNF5elZi_Gm-K9HhRilQXownlPrpjpc_mwWEeo0-r1-fLs2b9_vTNcrFuVMtoaTQFq8CA3thWaegZ6-WmY2bQpOPcDmRuwFKmJUBnNTeMDUbZHrQBboFSdoxe7H0vp81otKqnk_TiMrlRpisRpRP_boK7EF_idwG0r-egGjw9GKT4bTK5iNFlZbyXwcQpix54ywZKKvjkP3AbpxTqczsvwoF1uzjNHlIp5pyMvU5Cidg1KGqD4rrByj_-O_4NfaisAo_2wDaXmG72c6CMz9kfMp-hGg</recordid><startdate>20030106</startdate><enddate>20030106</enddate><creator>Farina, Kim L.</creator><creator>Hüttelmaier, Stefan</creator><creator>Musunuru, Kiran</creator><creator>Darnell, Robert</creator><creator>Singer, Robert H.</creator><general>Rockefeller University Press</general><general>The Rockefeller University Press</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QP</scope><scope>7QR</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20030106</creationdate><title>Two ZBP1 KH Domains Facilitate β-Actin mRNA Localization, Granule Formation, and Cytoskeletal Attachment</title><author>Farina, Kim L. ; Hüttelmaier, Stefan ; Musunuru, Kiran ; Darnell, Robert ; Singer, Robert H.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c431t-d12fc2e2dbf4cd27337ab53e8d0599f806e2f13da225fd9e338ecf72de29f2113</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2003</creationdate><topic>Actins</topic><topic>Actins - genetics</topic><topic>Actins - metabolism</topic><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Avian Proteins</topic><topic>Cell motility</topic><topic>Cell Movement</topic><topic>Cells</topic><topic>Chick Embryo</topic><topic>Cytoskeleton</topic><topic>Cytoskeleton - metabolism</topic><topic>Dose-Response Relationship, Drug</topic><topic>Fibroblasts</topic><topic>Gene Deletion</topic><topic>Glutathione Transferase - metabolism</topic><topic>In Situ Hybridization</topic><topic>In Situ Hybridization, Fluorescence</topic><topic>Messenger RNA</topic><topic>Microfilaments</topic><topic>Microscopy, Fluorescence</topic><topic>Molecular Sequence Data</topic><topic>Neurons</topic><topic>Neurotransmitters</topic><topic>Perceptual localization</topic><topic>Phenotype</topic><topic>Precipitin Tests</topic><topic>Protein Structure, Tertiary</topic><topic>Recombinant Proteins - metabolism</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA - metabolism</topic><topic>RNA, Messenger - metabolism</topic><topic>RNA-Binding Proteins - chemistry</topic><topic>RNA-Binding Proteins - metabolism</topic><topic>Sequence Homology, Amino Acid</topic><topic>Skeletal system</topic><topic>Transcription, Genetic</topic><topic>Transfection</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Farina, Kim L.</creatorcontrib><creatorcontrib>Hüttelmaier, Stefan</creatorcontrib><creatorcontrib>Musunuru, Kiran</creatorcontrib><creatorcontrib>Darnell, Robert</creatorcontrib><creatorcontrib>Singer, Robert H.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The Journal of cell biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Farina, Kim L.</au><au>Hüttelmaier, Stefan</au><au>Musunuru, Kiran</au><au>Darnell, Robert</au><au>Singer, Robert H.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Two ZBP1 KH Domains Facilitate β-Actin mRNA Localization, Granule Formation, and Cytoskeletal Attachment</atitle><jtitle>The Journal of cell biology</jtitle><addtitle>J Cell Biol</addtitle><date>2003-01-06</date><risdate>2003</risdate><volume>160</volume><issue>1</issue><spage>77</spage><epage>87</epage><pages>77-87</pages><issn>0021-9525</issn><eissn>1540-8140</eissn><coden>JCLBA3</coden><abstract>Chicken embryo fibroblasts (CEFs) localize β-actin mRNA to their lamellae, a process important for the maintenance of cell polarity and motility. The localization of β-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 β-actin mRNA. Overexpression of the four KH domains or certain subsets of these domains delocalized β-actin mRNA in CEFs and inhibited fibroblast motility, demonstrating the importance of ZBP1 function in both β-actin mRNA localization and cell motility.</abstract><cop>United States</cop><pub>Rockefeller University Press</pub><pmid>12507992</pmid><doi>10.1083/jcb.200206003</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | Actins Actins - genetics Actins - metabolism Amino Acid Sequence Animals Avian Proteins Cell motility Cell Movement Cells Chick Embryo Cytoskeleton Cytoskeleton - metabolism Dose-Response Relationship, Drug Fibroblasts Gene Deletion Glutathione Transferase - metabolism In Situ Hybridization In Situ Hybridization, Fluorescence Messenger RNA Microfilaments Microscopy, Fluorescence Molecular Sequence Data Neurons Neurotransmitters Perceptual localization Phenotype Precipitin Tests Protein Structure, Tertiary Recombinant Proteins - metabolism Reverse Transcriptase Polymerase Chain Reaction Ribonucleic acid RNA RNA - metabolism RNA, Messenger - metabolism RNA-Binding Proteins - chemistry RNA-Binding Proteins - metabolism Sequence Homology, Amino Acid Skeletal system Transcription, Genetic Transfection |
| title | Two ZBP1 KH Domains Facilitate β-Actin mRNA Localization, Granule Formation, and Cytoskeletal Attachment |
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