Real-Time Visualization of ZBP1 Association with β-Actin mRNA during Transcription and Localization

AbstractBackground: mRNA localization in somatic cells is an important mechanism for gene expression regulation. In fibroblasts, the protein ZBP1 associates with the sequence that localizes β-actin mRNA to the leading edge of fibroblasts, augmenting motility. β-actin mRNA localizes in a cytoskeleton-dependent manner, depending on intact actin and myosin ATP-hydrolysis, and is largely bound to the actin cytoskeleton. The ZBP1 protein contains four KH RNA binding domains and a classic RBD RNA binding domain. It also contains a putative nuclear import and export sequence, suggesting a nuclear phase in this protein's function.Results: Using high-speed imaging, we show here the targeting of this RNA binding protein to β-actin pre-mRNA transcripts in the nuclei of living cells and measure the residence time of the RNA-protein complex before it leaves the transcription site. Then, the RNA-protein particle is exported to the cytoplasm, where it localizes at velocities of 0.6 μm/s by using actin filaments and/or microtubules. This RNA-ZBP1 complex is required for cytoplasmic localization in fibroblasts; mislocalizing the protein also mislocalizes the RNA, and expressing the protein in a ZBP1-deficient cell line induces β-actin mRNA localization.Conclusions: This work demonstrates that the RNA-protein association, essential for cytoplasmic localization, begins as soon as the RNA is transcribed. The ZBP1 then forms a ribonucleoprotein particle and moves in a myosin-dependent fashion by using the cytoskeleton for directional transport