Mouse nucleolin binds to 4.5S RNA h, a small noncoding RNA

Abstract

4.5S RNA h is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNA h 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 RNA h-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 RNA h in vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNA h-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 RNA h recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNA h was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus.