The tripartite motif of nuclear factor 7 is required for its association with transcriptional units.
In amphibian oocytes, the maternal nuclear factor NF7 associates with the elongating pre-mRNAs present on the numerous lateral loops of the lampbrush chromosomes. Here, we have purified NF7 from an oocyte extract by using a combination of ion-exchange chromatography and gel filtration chromatography and demonstrated for the first time that nucleoplasmic NF7 exists primarily as free homotrimers. We confirmed the in vivo homotrimerization of NF7 by using a glutaraldehyde cross-linking assay, and we further showed that it only requires the coiled-coil domain of the NF7 tripartite motif/RBCC motif. Interestingly, we also obtained evidence that NF7 is recruited to the nucleus as a homotrimer, and expression of several mutated forms of NF7 in oocytes demonstrated that both the coiled coil and B box of NF7 are required for its chromosomal association. Together, these data strongly suggest that the interaction of NF7 with the active transcriptional units of RNA polymerase II is mediated by a trimeric B box. Finally, and in agreement with a role for NF7 in pre-mRNA maturation, we obtained evidence supporting the idea that NF7 associates with Cajal bodies.
Beenders
Brent
B
Department of Cell and Developmental Biology, School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Room B107 CLSL, Urbana, IL 61801, USA.
Jones
Peter Lawrence
PL
Bellini
Michel
M
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D002875:Chromosomes / Q000502:physiology; D020541:Coiled Bodies / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins; D004527:Egg Proteins; D005260:Female; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology*; D009865:Oocytes / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000502:physiology*; D012319:RNA Polymerase II / Q000235:genetics / Q000502:physiology; D012322:RNA Precursors / Q000235:genetics / Q000502:physiology; D029867:Xenopus Proteins / Q000737:chemistry / Q000502:physiology*; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004527:Egg Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D029867:Xenopus Proteins; C071551:XNF7 protein, Xenopus; D012319:RNA Polymerase II