Analysis of the molecular composition of Ro ribonucleoprotein complexes

Abstract

Human Ro ribonucleoproteins (RNPs) are composed of one of the four small Y RNAs and at least two proteins, Ro60 and La; association of additional proteins including the Ro52 protein and calreticulin has been suggested, but clear-cut evidence is still lacking. Partial purification of Ro RNPs from HeLa S100 extracts allowed characterization of several subpopulations of Ro RNPs with estimated molecular masses of between 150 and 550 kDa. The majority of these complexes contained Ro60 and La, whereas only a small proportion of Ro52 appeared to be associated with Ro RNPs. To identify novel Y RNA-associated proteins in vitro, binding of cytoplasmic proteins to biotinylated Y RNAs was investigated. In these reconstitution experiments, several proteins with estimated molecular masses of 80, 68, 65, 62, 60 and 53 kDa, the latter two being immunologically distinct from Ro60 and Ro52, respectively, appeared to bind specifically to Y RNAs. Furthermore, autoantibodies to these proteins were found in sera from patients with systemic lupus erythematosus. The proteins bound preferentially to Y1 and Y3 RNA but, with the exception of the 53-kDa protein, only weakly to Y4 RNA and not at all to Y5 RNA. Coprecipitation of the 80, 68, 65, and 53-kDa proteins by antibodies to Ro60 and La was observed, suggesting that at least a proportion of the novel proteins may reside on the same particles as La and/or Ro60. Finally, the binding sites for these proteins on Y1 RNA were clearly distinct from the Ro60-binding site involving a portion of the large central loop 2, which was found to be indispensable for binding of the 80, 68, 65 and 53-kDa proteins, as well as the stem 3±loop 3 and stem 2±loop 1 regions. Interestingly, truncation of the La-binding site resulted in decreased binding of the novel proteins (but not of Ro60), indicating La to be required for efficient association. Taken together, these results suggest the existence of further subpopulations of Ro RNPs or Y RNPs, consistent with the heterogeneous characteristics observed for these particles in the biochemical fractionation experiments.Keywords: autoantibodies; La; RNA±protein interactions; Ro52; Ro60.Ro ribonucleoproteins (RNPs), which may also be called Y RNPs, are composed of one of four small cytoplasmic RNAs (84±112 nucleotides) termed Y1±Y5 (Y2 is a degraded form of Y1 RNA) and at least two proteins: the 60-kDa protein, Ro60, and the 48-kDa phosphoprotein, La (reviewed in [1,2]). Ro60 and La belong to the family of RNA-binding proteins characterized by a conserved RNA-binding domain (also termed RNA-recognition motif ) of < 80 amino acids. A third protein of molecular mass 52 kDa (Ro52) has been reported to be associated with Ro RNPs, but this association has not been unequivocally proven so far [3±8]. Ro52 is completely unrelated to Ro60 and La and belongs to a group of proteins assumed to be involved in cell activation and transformation (reviewed in [9]). The four Y RNAs are closely related and are assumed to have similar predicted secondary structures. They are trans...

A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

“…Several proteins and RNPs have been reported to interact with this domain in different Y RNAs, including nucleolin, hnRNP K, hnRNP I, and 5S RNP (Fabini et al 2001;Fouraux et al 2002;Hogg and Collins 2007). These interactions do not occur with all Y RNAs (Fabini et al 2000(Fabini et al , 2001Fouraux et al 2002). This heterogeneity points toward additional specialized functions of individual Y RNAs mediated by this domain, which do not play a role in the cell-free DNA replication system.…”

APOBEC3G: an intracellular centurion

“…Ro RNPs are the major RNP autoantigens recognized by sera from patients with various connective tissue diseases. In human cells, Ro RNPs contain one of the four human small Y (hY ) RNAs (hY1, hY3, hY4 and hY5) and two core proteins (60 kDa Ro and 50 kDa La; Fabini et al 2000). Almost all of the proteins that participate in the formation of Staufen-containing RNA granules and Ro RNPs are readily detectable in the purified HMM A3G RNP complexes.…”

Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control

“…Proteins suggested to interact with Y RNPs include nucleolin and hnRNP I/PTB, both of which are reported to bind only Y1 and Y3 (Fabini et al 2000;Fouraux et al 2002), and Ro RNP-Binding Protein I (RoBPI/FIR/PUF60), a protein identified from diverse assays, including a yeast three-hybrid screen with the combination of Y5 RNA and Ro as bait (Page-McCaw et al 1999;Bouffard et al 2000;Liu et al 2000). To investigate the existence and specificity of substoichiometric interaction partners of an individual human Y RNA, we selected Y5 RNA for additional study.…”