Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains.
The IGF-II mRNA-binding proteins (IMPs), which are composed of two RNA recognition motifs, (RRM) and four hnRNP K homology (KH) domains, have been implicated in subcytoplasmic localization of mRNAs during embryogenesis. The IMP family originated via two gene duplications before the divergence of vertebrates, and IMP homologues consisting of only the four KH motifs have been identified in Drosophila and Caenorhabditis elegans. Here we characterise the trafficking of GFP-IMP1 fusion proteins and determine the structural determinants for proper cytoplasmic localization. GFP-IMP1 is present in large 200-700 nm RNP granules, which are distributed along microtubules. In motile cells, GFP-IMP1 is transported towards the leading edge into the cortical region of the lamellipodia where it is connected to microfilaments. Granules travel in an ATP-dependent fashion at an average speed of 0.12 microm/s (range 0.04-0.22 microm/s), and cells switch from a delocalized to a localized pattern within 15-20 minutes. Both granule formation and localization are unaffected by removal of the two RRMs, whereas deletion of the KH domains, which mediate RNA-binding, impairs these functions. We conclude that IMP1 localization is associated with motility and that the major functions of IMP1 are carried out by the phylogenetically conserved KH domains.
Nielsen
Finn C
FC
Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
Nielsen
Jacob
J
Kristensen
Mette A
MA
Koch
Grete
G
Christiansen
Jan
J
D016475:3T3 Cells; D000818:Animals; D003593:Cytoplasm / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins
C498777:Igf2bp3 protein, mouse; D008164:Luminescent Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D007335:Insulin-Like Growth Factor II