Characterization of a beta-actin mRNA zipcode-binding protein

Area of Science:

• Molecular Biology
• Cell Biology
• RNA Biology

Background:

• Beta-actin mRNA localization to fibroblast leading edges depends on a 54-nucleotide "zipcode" in its 3' untranslated region.
• Proteins interacting with the zipcode are hypothesized to mediate this crucial mRNA localization process.

Purpose of the Study:

• To identify proteins that bind to the beta-actin mRNA zipcode.
• To elucidate the role of these proteins in mRNA localization.

Main Methods:

• Band-shift mobility assays, UV cross-linking, and affinity purification were used to identify zipcode-binding proteins.
• Microsequencing, PCR amplification, and cDNA library screening were employed to clone and characterize the identified protein.
• Antipeptide antibodies were generated to study protein interactions and complex formation.

Main Results:

• A 68 kDa protein, ZBP-1, was identified that specifically binds to the proximal half of the zipcode.
• ZBP-1 shares homology with known RNA-binding proteins and possesses RNA recognition motifs and a putative nuclear export signal.
• ZBP-1 was found to function within a complex, co-immunoprecipitating with 120 and 25 kDa proteins, and affinity purification yielded 120 and 53 kDa proteins.
• Mutation of the ACACCC element within the zipcode significantly reduced protein binding and mRNA localization.

Conclusions:

• The 68 kDa ZBP-1 is an RNA-binding protein essential for beta-actin mRNA localization.
• ZBP-1 functions as part of a larger protein complex to mediate mRNA transport and localization in fibroblasts.