Real-time visualization of ZBP1 association with beta-actin mRNA during transcription and localization.
BACKGROUND
mRNA localization in somatic cells is an important mechanism for gene expression regulation. In fibroblasts, the protein ZBP1 associates with the sequence that localizes beta-actin mRNA to the leading edge of fibroblasts, augmenting motility. beta-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 beta-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 microm/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 beta-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.
Oleynikov
Yuri
Y
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Singer
Robert H
RH
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism; D030161:Avian Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002642:Chick Embryo; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
D000199:Actins; D000903:Antibiotics, Antineoplastic; D030161:Avian Proteins; D005231:Fatty Acids, Unsaturated; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C498636:ZBP1 protein, Gallus gallus; D009218:Myosins; C038753:leptomycin B