Abstract
The ribosomal protein L22 is a component of the 60S eukaryotic ribosomal subunit. As an RNA-binding protein, it has been shown to interact with both cellular and viral RNAs including 28S rRNA and the Epstein-Barr virus encoded RNA, EBER-1. L22 is localized to the cell nucleus where it accumulates in nucleoli. Although previous studies demonstrated that a specific amino acid sequence is required for nucleolar localization, the RNA-binding domain has not been identified. Here, we investigated the hypothesis that the nucleolar accumulation of L22 is linked to its ability to bind RNA. To address this hypothesis, mutated L22 proteins were generated to assess the contribution of specific amino acids to RNA binding and protein localization. Using RNA-protein binding assays, we demonstrate that basic amino acids 80–93 are required for high affinity binding of 28S rRNA and EBER-1 by L22. Fluorescence localization studies using GFP-tagged mutated L22 proteins further reveal that basic amino acids 80–93 are critical for nucleolar accumulation and for incorporation into ribosomes. Our data support the growing consensus that the nucleolar accumulation of ribosomal proteins may not be mediated by a defined localization signal, but rather by specific interaction with established nucleolar components such as rRNA.
Highlights
Of eukaryotic ribosomal subunits occurs in the cell nucleolus where ribosomal proteins are assembled along with rRNA by a myriad of processing and assembly factors
A 17-amino acid biotin acceptor peptide (BAP) was fused to the N-terminus of L22 and biotinylation was accomplished in vivo using a co-expressed bacterial biotin ligase (BirA). 293T cells were transiently co-transfected with expression constructs encoding BAP or BAP-L22, BirA, and EBER-1, EBER-2 or both EBERs
While EBER-1 and 28S rRNA were isolated along with biotinylated BAP-L22, EBER-2 was not isolated. None of these RNAs were isolated in the presence of only BAP demonstrating that the observed binding was specific for L22 and not an artifact of the BAP tag. These results clearly demonstrate that L22 binds strongly to full-length EBER-1 and, to a lesser extent, endogenous 28S rRNA in vivo
Summary
Of eukaryotic ribosomal subunits occurs in the cell nucleolus where ribosomal proteins are assembled along with rRNA by a myriad of processing and assembly factors (reviewed in: [1]). Ribosomal proteins, which like other proteins are translated in the cytoplasm, must be imported into the nucleus via an active transport mechanism mediated by a nuclear localization signal (NLS) and transit to the nucleolus. While many nucleolar proteins contain classical monopartite or bipartite NLSs [3,4], Stuger, et al proposed that eukaryotic ribosomal proteins utilize a unique nuclear import pathway mediated by a novel consensus NLS [5]. Because the nucleolus is not a membrane-bound structure, it is presumed that nucleolar accumulation occurs via interaction with established nucleolar components such as rRNA [2]. While a number of studies have examined the sequence requirements for the nucleolar localization of ribosomal proteins [7,8,9,10,11,12,13], relatively few have examined rRNA binding as a means for nucleolar accumulation [14,15,16,17]
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