Different Domains Cooperate to Target the Human Ribosomal L7a Protein to the Nucleus and to the Nucleoli

Abstract

The human ribosomal protein L7a is a component of the major ribosomal subunit. We transiently expressed in HeLa cells L7a-␤-galactosidase fusion proteins and studied their subcellular localization by indirect immunofluorescence staining with anti-␤-galactosidase antibodies. We have identified three distinct domains responsible for the nuclear targeting of the protein: domain I, amino acids 23-51; domain II, amino acids 52-100; domain III, amino acids 101-220, each of which contains at least one nuclear localization signal (NLS). Through subcellular localization analysis of deletion mutants of L7a-␤-galactosidase chimeras, we demonstrate that domain II plays a special role because it is necessary, although not sufficient, to target the chimeric ␤-galactosidase to the nucleoli. In fact, we demonstrate that the nucleolar targeting process requires the presence of domain II plus an additional basic domain that can be represented by an NLS or a basic stretch of amino acids without NLS activity. Thus, when multiple NLS are present, each NLS exerts distinct functions. Domain II drives nucleolar accumulation of a reporter protein with the cooperative action of a short basic amino acid sequence, suggesting a mechanism requiring protein-protein or protein-nucleic acid interactions.The biogenesis of eukaryotic ribosomes is a complex process that takes place in the cell nucleus. Soon after synthesis in the cytoplasm, ribosomal proteins (r-proteins) 1 are transported to the nucleus and subsequently accumulated in the nucleoli where they are associated with the precursor-rRNAs (pre-rRNAs), which are concomitantly processed into mature rRNA molecules (1). The assembled ribosomal subunits are eventually exported to the cytoplasm to function in protein biosynthesis. Thus, the biogenesis of eukaryotic ribosomes entails an intensive traffic of molecules across the nuclear membrane and, therefore, r-proteins are a good model with which to study the mechanism of nuclear transport and nucleolar accumulation of proteins. The nuclear transport of proteins depends upon the presence of one or more nuclear localization signals (NLS). These sequences have been found throughout the polypeptide chain (2) and, in most cases, consist of either short basic amino acid sequences like the NLS of the SV40 large T-antigen ( 126 PKKKRKV 132 ) (3, 4) or longer bipartite sequences consisting of two stretches of basic amino acids separated by about 10 amino acids (5). NLS are both necessary and sufficient to target a cytoplasmic protein to the nucleus (4). Much less is known about the mechanism of nucleolar targeting of proteins.Studies on the nucleolar accumulation of viral proteins have suggested that, like nuclear transport, nucleolar transfer is mediated by short amino acid sequences, namely nucleolar localization signals (NOS) (6 -9). A NOS motif, however, is not present in the cellular nucleolar proteins that have been identified so far, e.g. NO38 (10), nucleolin (11, 12), NSR1 (13), GAR1 (14). Studies on the targeting mechanism of the nucleolar p...

Nuclear and nucleolar targeting of human ribosomal protein S25: Common features shared with HIV-1 regulatory proteins

“…In S6, portions of approximately 60 residues were shown as minimum continuous fragments to confer nuclear/nucleolar targeting, in which two independent basic amino acid stretches seemed to cooperate (Schmidt et al, 1995). In the case of L7a, the amino-terminal 100 residue fragment was necessary and sucient for the nuclear/nucleolar targeting, which was also enabled by the combination of 17 residue amino terminal peptide and another portion (residue number 52 ± 100) in the fragment (Russo et al, 1997). However, most interestingly, several small and distinct NOSs, which are similar to the NLS/NOS of RPS25, have been identi®ed in retroviral RNA-binding trans-regulator proteins.…”

“…Such a compact NOS is not commonly found in eukaryotic cellular proteins. More generally, nucleolar localizing properties seem to be acquired by cooperation of several functional domains (Russo et al, 1997;Peculis and Gall, 1992;SchmidtZachmann and Nigg, 1993;Yan and Me leÁ se, 1993;Michael and Dreyfuss, 1996). Among these proteins, no common or prototypic NOS motif has been presented, suggesting the complexity of the nucleolar localizing activity.…”

“…Indeed, all the recent similar works on human ribosomal proteins have employed either epitopetagging systems (Moreland et al, 1985;Michael and Dreyfuss, 1996;Martin-Nieto and Roufa, 1997) or even b-galactosidase fusions (Schmidt et al, 1995;Russo et al, 1997). Following the more well-accepted former strategy, an RPS25 expression vector, pcFL-RPS25, was designed ( Figure 1a).…”

“…The functional signi®cance of the nucleolar localization of these proteins is still controversial, although a possible role of the nucleolus as a storage site for the proteins is suggested in the case of the HIV-1 Rev protein (Kubota et al, 1996), which shuttles between the cytoplasm and the nucleus, to enable the nuclear export of unspliced viral mRNAs (Malim et al, 1989a,b;Fischer et al, 1994;Kalland et al, 1994;Meyer and Malim, 1994;Richard et al, 1994). Despite its evident functional signi®cance as a pre-event of ribosomal assembly, nucleolar targeting of human ribosomal proteins has been characterized in only a few members, S6, S14 and L7a (Schmidt et al, 1995;Martin-Nieto and Roufa, 1997;Russo et al, 1997). Therefore, further analyses of the nucleolar targeting of other ribosomal proteins seem to be required to gain insights into not only ribosomal biogenesis, but also the role of the nucleolar localization of such RNAbinding transactivators.…”

Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene

“…These include: neurotropic tyrosin receptor kinase 2 gene (NTRK2, also called TRKB) that belongs to a gene family involved in cell survival, growth and differentiation and in tumor progression (Shibayama and Koizumi, 1996;Sugimoto et al, 2001); IL-1R-associated kinase 2 (IRAK2) gene that encodes for a protein involved in Ras-dependent cell signalling (Palsson et al, 2002); p42 mitogen-activated protein kinase 1 (p42MAPK1) gene, that encodes for a protein involved in cell signalling (Ras/MAPK pathway) and is hyperexpressed in medulloblastoma (MacDonald et al, 2001); inositol 1,4,5-triphosphate receptor type 1 (IP3R1) gene and inositol 1,4,5-triphosphate receptor type 2 (IP3R2) gene that encode for calcium channels located in the endoplasmic reticulum (ER) membrane (releasing calcium from the ER) (Pozzan et al, 1994) and that are involved in cell transformation and progression (Weber et al, 1996); alpha 2,3 sialyltransferase gene (called SITA or ST3GalVI), involved in protein glycosylation and tumor invasion (Petretti et al, 2000;Schneider et al, 2001;Zhu et al, 2001); thyroid hormone uncoupling protein gene (TRUP), also called ribosomal protein L7a, encoding for a protein that interacts with thyroid hormone receptor and interferes with the ability of the thyroid hormone and retinoic acid receptors to interact with DNA (Burris et al, 1995;Russo et al, 1997), and whose expression has been involved in human tumors (Kroes et al, 2000;Wang et al, 2000); EMX2-like gene, a Genscan predicted gene containing a homeobox homologous domain, the encoded protein of which is 70% homologous (56% identical) with the human homeobox protein EMX2 (Swissprot Accession number Q04743) (homeobox containing genes encode for transcription factors involved in development) and human telomerase reverse transcriptase (hTERT) gene, involved in cell immortalization and expressed in the majority of human malignancies (Hanahan and Weinberg, 2000;Meyerson, 2000).…”