Association of guanine nucleotide-exchange protein BIG1 in HepG2 cell nuclei with nucleolin, U3 snoRNA, and fibrillarin
2008
Padilla, Philip Ian | Uhart, Marina | Pacheco-Rodriguez, Gustavo | Peculis, Brenda A. | Moss, Joel | Vaughan, Martha
BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, activates class I ADP-ribosylation factors (ARF1-3) by catalyzing the replacement of bound GDP by GTP, an action critical for the regulation of protein transport in eukaryotic cells. Our earlier report [Padilla PI, Pancheco-Rodriguez G, Moss J, Vaughan M (2004) Proc Natl Acad Sci USA 101:2752-2757] that BIG1 concentrated in nucleoli of serum-starved HepG2 cells prompted us to identify molecules associated with BIG1 in dynamic nucleolar structures. Antibodies against BIG1 or nucleolin coprecipitated both proteins from nuclei, which was abolished by the incubation of nuclei with RNase A or DNase, indicating that the interaction depended on nucleic acids. ³²P labeling of RNAs immunoprecipitated with BIG1 or nucleolin from nuclei revealed bands of [almost equal to]210 bases that also hybridized with U3 small nucleolar (sno)RNA-specific oligonucleotides. Clones of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or nucleolin yielded identical nucleotide sequences that also were found in genomic DNA. Later analyses revealed the presence of fibrillarin, nucleoporin p62, and La in BIG1 and nucleolin immunoprecipitates. Our data demonstrate that BIG1, nucleolin, U3, the U3-binding protein fibrillarin, and the RNA-binding protein La may exist together in nuclear complexes, consistent with a potential role for BIG1 in nucleolar processes. Evidence that BIG1 and nucleolin, but not fibrillarin, can be present with p62 at the nuclear envelope confirms the presence of BIG1 and nucleolin in dynamic molecular complexes that change in composition while moving through nuclei. Nuclear functions of BIG1 remain to be determined.
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