Molecular composition of Ro small ribonucleoprotein complexes in human cells. Intracellular localization of the 60- and 52-kD proteins.

Abstract

Ro small ribonucleoprotein complexes (RoRNPs) are thought to comprise several proteins, including the 60-kD Ro and the 52-kD Ro proteins, and several small RNAs, designated Y RNAs. Although RoRNPs are fairly ubiquitous in nature, their precise composition remains unknown, their function has been elusive, and their intracellular localization has been controversial. We have analyzed HeLa cell extracts by glycerol density gradient fractionation in order to determine the distribution of the individual protein and RNA components of RoRNPs. We found that 52-kD Ro was not detectable in an RNP complex with the 60-kD protein under a variety of conditions. Pretreatment of cell extracts with ribonuclease affected gradient migration of the 60-kD but not the 52-kD protein, suggesting that the latter is not complexed with RNA. The migration of the hY RNAs in these gradients closely followed that of 60-kD and not 52-kD Ro. Immunofluorescence analysis of two different cell lines with monospecific antibodies against 52-and 60-kD proteins strongly suggests that these two proteins are not present on overlapping sets of structures in vivo. We conclude that the 52-kD Ro protein is not a detectable component of the RoRNP complex under these conditions despite its reactivity with Ro autoimmune antisera. (J. Clin. Invest. 1994. 93:1637-1644

RNA-labelled Ro and La ribonucleoprotein complexes reassembled in vitro; characterization by gel shift analysis

“…Rabbit anti-52-kD Ro antibodies failed to retard any of the three complexes. This suggested that the 52-kD protein is not part of the reassembled complexes, in line with recent biochemically obtained data suggesting that the 52-kD protein is not part of native Ro RNPs [8,9].…”

Production of anti-Ro/SS-A and anti-La/SS-B Autoantibodies is Closely Coordinated in Systemic Lupus Erythematosus and Independent of anti-dsDNA Production

“…Recent murine studies have convincingly demonstrated that anti-Ro/SS-A and anti-La/SS-B antibodies can be the product of immunization with one particle, a ribonucleoprotein particle containing both 60 kDa Ro/SS-A and 50 kDa La/SS-B proteins coupled to yRNA molecules [20]. Another (52 kDa) Ro/SS-A protein exists which is probably not a constituent of the scRNP particles [34]. There is some resemblance between the above studies and those by Mamula and coworkers, who elegantly demonstrated the prerequisites for the induction of true autoantibodies against the different small nuclear ribonucleoproteins (snRNPs) in immunized mice [18,35].…”

Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins

“…Coprecipitation of RoBPI and Ro RNPs from HeLa cell extracts using anti-Ro60 antibodies+ HeLa cells were incubated on ice with DTSSP alone without treatment with digitonin (untreated; A), permeabilized with digitonin but without crosslinking (B), permeabilized with digitonin and crosslinked with DTSSP (C), before immunoprecipitation of soluble extracts using a human anti-Ro60 serum (the same used in immunoblot in lane Ro60 of each panel)+ Immunoprecipitated proteins were run in SDS-PAGE, electrotransferred to nitrocellulose, and blotted using antisera against Ro60 (lane Ro60), Ro60/La (lane Ro/La), and PUF (lane RoBP1)+ As a molecular weight control, total HeLa cell extracts were run in parallel (D) and blotted with the same antisera+ In addition to the 65-kDa band corresponding to RoBPI, chicken anti-PUF serum also decorated other antigens at about 40-50 kDa present in cell extracts and in immunoprecipitates from anti-Ro sera and from other autoimmune sera (data not shown)+ These antigens were not further defined, as they were not recognized by RoBPI antisera (see Fig+ 6)+ FIGURE 6. Recognition of recombinant and native RoBPI by anti-PUF antibodies and RoBPI antisera+ His-tagged recombinant RoBPI (A) and native RoBPI from crude HeLa cell extracts (B) were separated in 10% SDS-PAGE and electrotransferred to nitrocellulose+ The proteins were incubated with antisera and bound antibodies detected by chemiluminescence+ Lane 0: pre-immune rabbit serum+ Lane 1: anti-PUF antibodies+ Lanes 2-4: RoBPI antisera from three different rabbits immunized with RoBPI+ Anti-PUF antibodies and RoBPI antisera recognized the same protein bands+ A 52-kDa protein dubbed Ro52 was previously reported as a component of Ro RNPs (Ben-Chetrit et al+, 1988)+ Denatured Ro52 is recognized in immunoblot by antibodies present in most anti-Ro sera+ Recognition of the native protein is far more controversial, however, because Ro52 is not immunoprecipitated by anti-Ro sera from 35 S-labeled cell extracts (Buyon et al+, 1994), even after prolonged (40 h) labeling of exponentially growing cells (data not shown)+ Although there is indirect immunological evidence of an association of Ro52 with Ro60 or Ro RNPs (Slobbe et al+, 1992;Peek et al+, 1994;Topfer et al+, 1995;Tseng et al+, 1997), no direct biochemical demonstration of an association has been provided using either classical (Kelekar et al+, 1994;Boire et al+, 1995) or yeast interaction assays (authors' unpubl+ data)+ RoBPI thus stands distinctly as the first bona fide Ro RNP partner+ Ro60 was recently shown to be an essential cofactor for the alternative interaction of La and cellular nucleic acid binding protein (CNBP) with the 59 UTR of X. laevis ribosomal protein mRNAs, affecting the translational fate of these mRNAs (Pellizzoni et al+, 1998)+ Ro60, as well as an unidentified RNA component, was essential for binding of La and CNBP to specific regions of the 59 UTR+ It remains unknown whether Ro60 directly interacts with La or CNBP proteins or with ribosomal protein mRNAs, or whether Ro60 is part of a macromolecular complex implicated in the alternative binding of La and CNBP+ It is also currently unknown whether Y RNAs are implicated in the activation of the cofactor, acting as a putative link between Ro60 and La+ One has to recall that mature Xenopus Y RNAs found in Ro RNPs have no 39 oligouridylate extension that would allow binding of La+ If Y RNAs are indeed part of the requi...…”

“…Ro RNPs are low-copy RNPs present in all mammalian cells, including mature (anuclear) human erythrocytes (Rader et al+, 1989) and platelets (Itoh & Reichlin, 1991)+ Ro RNP homologs are also found in Caenorhabditis elegans (Labbé et al+, 1995;van Horn et al+, 1995) and Xenopus laevis (O'Brien et al+, 1993)+ Y RNAs, the nucleic acid component of Ro RNPs, are small noncoding RNAs transcribed by RNA polymerase (pol) III (Hendrick et al+, 1981)+ The size and number of RNAs of the Y family vary somewhat between cell types and species (Mamula et al+, 1989a;Rader et al+, 1989;Itoh & Reichlin, 1991)+ The subcellular localization of Ro RNPs is controversial (Hendrick et al+, 1981;Ben-Chetrit et al+, 1988;Peek et al+, 1993;Kelekar et al+, 1994)+ In all species, a moderately conserved 60-kDa (Ro60) protein binds to Y RNAs by interacting with a highly conserved duplex structure resulting from base pairing between their 59 and 39 ends (Wolin & Steitz, 1984)+ In humans, Ro RNPs comprise one of the four hY RNAs (hY1, hY3, hY4, or hY5) associated with Ro60 and, at least in a fraction of Ro RNPs, with a 48-kDa protein called La (Mamula et al+, 1989b;Boire & Craft, 1990)+ The La protein binds to the 39 oligouridylate end of RNA pol III transcripts (Stefano, 1984;Slobbe et al+, 1992) and is an RNA pol III transcription termination factor (Gottlieb & Steitz, 1989)+ La protein also plays a role in transcript release, reinitiation, and even processing of nascent transcripts (Maraia, 1996;Goodier et al+, 1997;van Horn et al+, 1997;Fan et al+, 1998), and is implicated in the regulation of translation of some viral (Meerovitch et al+, 1993;Chang et al+, 1994;Svitkin et al+, 1994;Ali & Siddiqui, 1997) and cellular (Pellizzoni et al+, 1996(Pellizzoni et al+, , 1998 mRNAs+ Both the oligouridylate tail and the associated La binding are usually lost during maturation of pol III transcripts …”