The RoRNP complex in mammalian cells was first described as being composed of one of at least four hY RNAs associated with two proteins of 52 kDa (Ro/SSA52) and 60 kDa (Ro/SSA60) (Ben-Chetrit et al 1988, Rader et al. 1989). hY RNAs are small RNAs known as hY1, hY2, hY3, hY4, and hY5 RNA ranging from 84 to 112 nucleotides and are present in about 1 to 5x105 copies per cell (Wolin and Steitz 1984).All Y RNAs can be folded into a structure containing a large internal loop and a long stem formed by base pairing the 5' and 3' ends (Van Horn et al. 1995). Within the stem is a highly conserved bulged helix that is the binding site for the Ro/SSA protein (Green et al. 1998). In human cells, RoRNPs consist of one of the four hY RNAs and two core proteins: Ro/SSA60 and La/SSB. Physicochemical studies on native RoRNPs indicate that the particles segregate into three discrete subpopulations, one containing hY5, another containing hY4, and a third containing hY1, hY3, and hY4 (Boire and Craft 1989). Initially, it seemed that the 60-kDa Ro/SSA polypeptide was the exclusive protein component in the complex, but Boire and Craft (Boire and Craft 1990) were able to biochemically isolate RoRNPs in which the autoantigen La/SSB was a stable component.
Taken together, biochemical characterization of the RoRNP complex lead to the following model: the hY RNA is noncovalently bound to the 60-kDa Ro/SSA via the lower stem of the RNA formed by base pairing their 5' and 3' ends (Wolin and Steitz 1984), whereas La/SSB binds to the 3' oligo uridylate residues of hY RNAs (Fig. 24.2) (van Venrooij et al. 1993). In addition to the two core proteins, conditional association of other proteins such as calreticulin (Rokeach et al. 1991), Ro/SSA52 (Slobbe et al. 1992), RoBPI, and the RNA-binding protein nucleolin (Fournaux et al. 2002) to the RoRNP complex has been reported. However, these associations seem to be dependent on the structure of the hY RNA.
Although the evolutionarily conserved RoRNPs have been the subject of investigation in a number of laboratories for many years, their precise molecular definition, function, and subcellular localization remained unclear. The cellular distribution of Ro/SSA has been a matter of controversy since the early description of the autoantigen, because the Ro/SSA antigen seems to be present in both the cytoplasm and the nucleus. Although reports on association of Ro with cytoplasmic hY RNAs suggested
Fig. 24.2. The structure of the Ro ribonucleoprotein (RoRNP) particle (schematic diagram). In most higher eukaryotic cells, the Ro60 kDa protein is complexed with one of several small cytoplasmic RNAs known as Y RNAs. The Y RNA is folded into a structure containing internal loops and a long stem formed by base pairing the 5' and 3' ends. The 3' end of Y RNA is stably associated with the La protein, whereas the association of Ro52, nucleolin, RoBPI, and calreticulin is conditional
Fig. 24.2. The structure of the Ro ribonucleoprotein (RoRNP) particle (schematic diagram). In most higher eukaryotic cells, the Ro60 kDa protein is complexed with one of several small cytoplasmic RNAs known as Y RNAs. The Y RNA is folded into a structure containing internal loops and a long stem formed by base pairing the 5' and 3' ends. The 3' end of Y RNA is stably associated with the La protein, whereas the association of Ro52, nucleolin, RoBPI, and calreticulin is conditional that RoRNPs are cytoplasmic RNP particles (Lerner et al. 1981, Peek et al. 1993), the study of Harmon et al. (Harmon et al. 1984) (see also Fig. 24.1) clearly showed that specific Ro/SSA antibodies predominantly detect nucleoplasmic speckles. Corroborating the idea of nuclear Ro/SSA location, rabbit anti-Ro/SSA60 antibodies displayed a speckled nucleoplasmic immunofluorescence staining pattern, which could be completely abolished by addition of purified Ro/SSA60 protein in serologic competition experiments (Mamula et al. 1989). Moreover, Ro/SSA52 contains transcription factor-like motifs, for example, leucine zippers, which have been shown to promote protein dimer formation and to contribute to inhibition of transcriptional activity in vitro (Wang et al. 2001).
Cloning of complementary DNAs (cDNAs) for Ro/SSA allowed further characterization of the proteins. The complete cDNA and protein sequence of Ro/SSA52 (Gen-Bank/EBML accession numbers M35041) revealed that the open reading frame consists of 475 amino acid residues with a predicted molecular mass of 54 Kda and pi of 6.35. Based on the amino acid sequence, Ro/SSA52 is expected to contain three domains: (a) an N-terminal zinc finger domain, (b) a central coiled-coil (leucine zipper) domain, and (c) a C-terminal rfp-like domain. The N-terminal zinc finger domain is a member of the previously described 'Ring' finger proteins (Reddy et al. 1992), and the C-terminal rfp-like domain belongs to a second protein domain super-family named after the rfp protein (Takahashi et al. 1988). It is important to note that all sequence motifs of Ro/SSA52 represent DNA and/or RNA-binding motifs. The full-length cDNA sequences of the 60-kDa Ro/SSA protein has been published by Deutscher et al. (Deutscher et al. 1988) and Ben-Chetrit et al. (Ben-Chetrit et al. 1989) and showed identical sequences except the C-terminal nucleotides. Ro/SSA60, like Ro/SSA52, contains nucleic acid binding motifs. The N-terminal RNA binding domain is highly conserved between Xenopus and human Ro/SSA60 sequences (O'Brian et al. 1993) and may account for the binding of the 60-kDa protein to hY RNAs. In contrast, the function of the Ro/SSA60 zinc finger domain has not yet been characterized.
The genes for Ro/SSA52 and Ro/SSA60 have not been published yet. However, using cDNA probes it was demonstrated that the human Ro/SSA52 gene is located on chromosome 11 (Frank et al. 1993), and the human Ro/SSA60 gene has been localized to gene locus 1q31 by fluorescence in situ hybridization (Chan et al. 1994).
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