Characterization of a β-actin mRNA zipcode-binding protein

Anthony F. Ross, Yuri Oleynikov, Edward H. Kislauskis, Krishan L. Taneja, Robert H. Singer

Research output: Contribution to journalArticle

435 Citations (Scopus)

Abstract

Localization of β-actin mRNA to the leading edge of fibroblasts requires the presence of conserved elements in the 3' untranslated region of the mRNA, including a 54-nucleotide element which has been termed the 'zipcode' (E. Kislauskis, X. Zhu, and R. H. Singer, J. Cell Biol. 127:441- 451, 1994). In order to identify proteins which bind to the zipcode and possibly play a role in localization, we performed band-shift mobility assays, UV cross-linking, and affinity purification experiments. A protein of 68 kDa was identified which binds to the proximal (to the coding region) half of the zipcode with high specificity (ZBP-1). Microsequencing provided unique peptide sequences of approximately 15 residues each. Degenerate primers corresponding to the codons derived from the peptides were synthesized and used for PCR amplification. Screening of a chicken cDNA library resulted in isolation of several clones providing a DNA sequence encoding a 67.7-kDa protein with regions homologous to several RNA-binding proteins, such as hnRNP E1 and E2, and with consensus mRNA recognition motif with RNP1 and 2 motifs and a putative REV-like nuclear export signal. Antipeptide antibodies were raised in rabbits which bound to ZBP-1 and coimmunuprecipitated proteins of 120 and 25 kDa. The 120-kDa protein was also obtained by affinity purification with the RNA zipcode sequence, along with a 53-kDa protein, but the 25-kDa protein appeared only in immunoprecipitations. Mutation of one of the conserved sequences within the zipcode, an ACACCC element in its proximal half, greatly reduced its protein binding and localization properties. These data suggest that the 68-kDa ZBP-1 we have isolated and cloned is an RNA- binding protein that functions within a complex to localize β-actin mRNA.

Original languageEnglish (US)
Pages (from-to)2158-2165
Number of pages8
JournalMolecular and Cellular Biology
Volume17
Issue number4
StatePublished - Apr 1997

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Actins
Carrier Proteins
Messenger RNA
Proteins
RNA-Binding Proteins
Nuclear Export Signals
Heterogeneous-Nuclear Ribonucleoproteins
Peptides
Conserved Sequence
3' Untranslated Regions
Electrophoretic Mobility Shift Assay
Gene Library
Immunoprecipitation
Protein Binding
Codon
Chickens
Nucleotides
Clone Cells
Fibroblasts
Rabbits

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Ross, A. F., Oleynikov, Y., Kislauskis, E. H., Taneja, K. L., & Singer, R. H. (1997). Characterization of a β-actin mRNA zipcode-binding protein. Molecular and Cellular Biology, 17(4), 2158-2165.

Characterization of a β-actin mRNA zipcode-binding protein. / Ross, Anthony F.; Oleynikov, Yuri; Kislauskis, Edward H.; Taneja, Krishan L.; Singer, Robert H.

In: Molecular and Cellular Biology, Vol. 17, No. 4, 04.1997, p. 2158-2165.

Research output: Contribution to journalArticle

Ross, AF, Oleynikov, Y, Kislauskis, EH, Taneja, KL & Singer, RH 1997, 'Characterization of a β-actin mRNA zipcode-binding protein', Molecular and Cellular Biology, vol. 17, no. 4, pp. 2158-2165.
Ross AF, Oleynikov Y, Kislauskis EH, Taneja KL, Singer RH. Characterization of a β-actin mRNA zipcode-binding protein. Molecular and Cellular Biology. 1997 Apr;17(4):2158-2165.
Ross, Anthony F. ; Oleynikov, Yuri ; Kislauskis, Edward H. ; Taneja, Krishan L. ; Singer, Robert H. / Characterization of a β-actin mRNA zipcode-binding protein. In: Molecular and Cellular Biology. 1997 ; Vol. 17, No. 4. pp. 2158-2165.
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