Mouse nucleolin binds to 4.5S RNAh, a small noncoding RNA.
4.5S RNAh is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNAh is known to associate with cellular poly(A)-terminated RNAs and retroviral genomic RNAs, its function remains unclear. In this study, we analyzed 4.5S RNAh-binding proteins in mouse nuclear extracts using gel mobility shift and RNA-protein UV cross-linking assays. We found that at least nine distinct polypeptides (p170, p110, p93, p70, p48, p40, p34, p20, and p16.5) specifically interacted with 4.5S RNAhin vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNAh-binding proteins, we performed expression cloning from a mouse cDNA library and obtained cDNA clones derived from nucleolin mRNA. We identified p110 as nucleolin using nucleolin-specific antibodies. UV cross-linking analysis using various deletion mutants of nucleolin indicated that the third of four tandem RNA recognition motifs is a major determinant for 4.5S RNAh recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNAh was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus.
Hirose
Yutaka
Y
Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. yh620@pha.u-toyama.ac.jp
Harada
Fumio
F
D000818:Animals; D001665:Binding Sites; D002466:Cell Nucleolus / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015723:Gene Library; D047468:Immunoprecipitation; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D022661:RNA, Untranslated / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000097796:Nucleolin
D009687:Nuclear Proteins; D010750:Phosphoproteins; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D000097796:Nucleolin