Analysis of the intracellular localization and assembly of Ro ribonucleoprotein particles by microinjection into Xenopus laevis oocytes

Xenopus laevis oocytes have been used to determine the intracellular localization of components of Ro ribonucleoprotein particles (Ro RNPs) and to study the assembly of these RNA-protein complexes. Microinjection of the protein components of human Ro RNPs, i.e., La, Ro60, and Ro52, in X. laevis oocytes showed that all three proteins are able to enter the nucleus, albeit with different efficiencies. In contrast, the RNA components of human Ro RNPs (the Y RNAs) accumulate in the X. laevis cytoplasm upon injection. Localization studies performed at low temperatures indicated that both nuclear import of Ro RNP proteins and nuclear export of Y RNAs are mediated by active transport mechanisms. Immunoprecipitation experiments using monospecific anti-La and anti-Ro60 antibodies showed that the X. laevis La and Ro60 homologues were cross-reactive with the respective antibodies and that both X. laevis proteins were able to interact with human Y1 RNA. Further analyses indicated that: (a) association of X. laevis La and Ro60 with Y RNAs most likely takes place in the nucleus; (b) once formed, Ro RNPs are rapidly exported out of the nucleus; and (c) the association with La is lost during or shortly after nuclear export.     

Human monoclonal anti-La antibodies. The La protein resides on a subset of Ro particles

We have addressed the problem of anti-La autoimmune responses by defining the specific binding sites of human mAb to the La protein. Two human anti-La mAb were developed; one an IgM (kappa) (designated 8G3) and the second an IgG1 (kappa) (9A5) isotype. The mAb 8G3 immunoprecipitated the La RNA and La protein from crude human cell lysates; bound the 50-kDa La protein and a 28-kDa digestion fragment in immunoblots, and recognized a small defined internal segment from the cloned La protein. In contrast, the IgG isotype (9A5) failed to precipitate native La from cell lysates but bound the same segment of digested La protein and the same polypeptide of 131 amino acids in length from the cloned La protein. Immunoprecipitation experiments performed with these mAb demonstrated that the La protein is a component of a subset of Ro particles. The data suggest that the La protein is not present on the hY RNA in the absence of the Ro polypeptide. These observations may define functional subsets or maturation states of hY RNA based on their association with Ro or Ro and La polypeptides.     

Analysis of the molecular composition of Ro ribonucleoprotein complexes. Identification of novel Y RNA-binding proteins.

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.     

Comparison of cellular ribonucleoprotein complexes associated with the APOBEC3F and APOBEC3G antiviral proteins

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3F (APOBEC3F [A3F]) and A3G proteins are effective inhibitors of infection by various retroelements and share approximately 50% amino acid sequence identity. We therefore undertook comparative analyses of the protein and RNA compositions of A3F- and A3G-associated ribonucleoprotein complexes (RNPs). Like A3G, A3F is found associated with a complex array of cytoplasmic RNPs and can accumulate in RNA-rich cytoplasmic microdomains known as mRNA processing bodies or stress granules. While A3F RNPs display greater resistance to disruption by RNase digestion, the major protein difference is the absence of the Ro60 and La autoantigens. Consistent with this, A3F RNPs also lack a number of small polymerase III RNAs, including the RoRNP-associated Y RNAs, as well as 7SL RNA. Alu RNA is, however, present in A3F and A3G RNPs, and both proteins suppress Alu element retrotransposition. Thus, we define a number of subtle differences between the RNPs associated with A3F and A3G and speculate that these contribute to functional differences that have been described for these proteins.     

Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA).

We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA. The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA.     

Analysis of protein--RNA interactions within Ro ribonucleoprotein complexes.

The interactions between Ro and La proteins and hY RNAs have been analysed. The binding site for the 60 kDa Ro protein on hY RNAs is shown to be the terminal part of the base paired stem structure, which contains the most highly conserved sequence among hY RNAs. The bulged C-residue within this region plays an important role in the recognition by this protein. The same regions of hY RNAs are essential for the association of the 52 kDa Ro protein with the RNAs, strongly suggesting that the 60 kDa Ro protein is required for the 52 kDa Ro protein to bind, presumably via protein-protein interactions, to Ro RNPs. The binding site for the La protein on hY RNAs is shown to be the oligouridylate stretch near the 3'-end of the RNAs, which is also recognized when additional nucleotides flank this motif at the 3'-side. Additional sequence elements in hY3 and hY5, but not in hY1, are bound by the La protein as well. Deletion mutagenesis showed that the RNP motif, previously identified in many ribonucleoprotein (RNP) proteins and in some cases shown to be almost sufficient for the interaction with RNA, of both the 60 kDa Ro and the La protein are not sufficient for the interaction with hY RNAs. Substantial parts of these proteins flanking the RNP motif are needed as well. It is likely that they stabilize the correct conformation of the RNP motif for RNA binding.     

The heterogeneous nuclear ribonucleoproteins I and K interact with a subset of the ro ribonucleoprotein-associated Y RNAs in vitro and in vivo

The hY RNAs are a group of four small cytoplasmic RNAs of unknown function that are stably associated with at least two proteins, Ro60 and La, to form Ro ribonucleoprotein complexes. Here we show that the heterogeneous nuclear ribonucleoproteins (hnRNP) I and K are able to associate with a subset of hY RNAs in vitro and demonstrate these interactions to occur also in vivo in a yeast three-hybrid system. Experiments performed in vitro and in vivo with deletion mutants of hY1 RNA revealed its pyrimidine-rich central loop to be involved in interactions with both hnRNP I and K and clearly showed their binding sites to be different from the Ro60 binding site. Both hY1 and hY3 RNAs coprecipitated with hnRNP I in immunoprecipitation experiments performed with HeLa S100 extracts and cell extracts from COS-1 cells transiently transfected with VSV-G-tagged hnRNP-I, respectively. Furthermore, both anti-Ro60 and anti-La antibodies coprecipitated hnRNP I, whereas coprecipitation of hnRNP K was not observed. Taken together, these data strongly suggest that hnRNP I is a stable component of a subpopulation of Ro RNPs, whereas hnRNP K may be transiently bound or interact only with (rare) Y RNAs that are devoid of Ro60 and La. Given that functions related to translation regulation have been assigned to both proteins and also to La, our findings may provide novel clues toward understanding the role of Y RNAs and their respective RNP complexes.     

The interactions with Ro60 and La differentially affect nuclear export of hY1 RNA.

Ro RNPs are evolutionarily conserved ribonucleoprotein particles that consist of a small RNA, known as Y RNA, associated with several proteins, such as La, Ro60, and Ro52. The Y RNAs (Y1-Y5), which are transcribed by RNA polymerase III, have been shown to reside almost exclusively in the cytoplasm as Ro RNPs. To obtain more insight into the nuclear export pathway of Y RNAs, hY1 RNA export was studied in Xenopus laevis oocytes. Injection of various hY1 RNA mutants showed that an intact Ro60 binding site is a prerequisite for nuclear export, whereas the presence of an intact La binding site resulted in strong nuclear retention of hY1 RNA. Competition studies with various classes of RNAs indicated that, in addition to Ro60, another titratable factor was necessary for nuclear export of hY1 RNA. This factor appears also to be involved in nuclear export of tRNA. Because export of hY1 RNA could not be blocked by a synthetic peptide containing the recently identified nuclear export signal of the HIV-1 Rev protein, nuclear export of hY1 RNA does not seem to be dependent on a Rev-like nuclear export signal.     

Ribonucleoprotein particles containing non-coding Y RNAs, Ro60, La and nucleolin are not required for Y RNA function in DNA replication

Background

Ro ribonucleoprotein particles (Ro RNPs) consist of a non-coding Y RNA bound by Ro60, La and possibly other proteins. The physiological function of Ro RNPs is controversial as divergent functions have been reported for its different constituents. We have recently shown that Y RNAs are essential for the initiation of mammalian chromosomal DNA replication, whereas Ro RNPs are implicated in RNA stability and RNA quality control. Therefore, we investigate here the functional consequences of RNP formation between Ro60, La and nucleolin proteins with hY RNAs for human chromosomal DNA replication.

Methodology/Principal Findings

We first immunoprecipitated Ro60, La and nucleolin together with associated hY RNAs from HeLa cytosolic cell extract, and analysed the protein and RNA compositions of these precipitated RNPs by Western blotting and quantitative RT-PCR. We found that Y RNAs exist in several RNP complexes. One RNP comprises Ro60, La and hY RNA, and a different RNP comprises nucleolin and hY RNA. In addition about 50% of the Y RNAs in the extract are present outside of these two RNPs. Next, we immunodepleted these RNP complexes from the cytosolic extract and tested the ability of the depleted extracts to reconstitute DNA replication in a human cell-free system. We found that depletion of these RNP complexes from the cytosolic extract does not inhibit DNA replication in vitro. Finally, we tested if an excess of recombinant pure Ro or La protein inhibits Y RNA-dependent DNA replication in this cell-free system. We found that Ro60 and La proteins do not inhibit DNA replication in vitro.

Conclusions/Significance

We conclude that RNPs containing hY RNAs and Ro60, La or nucleolin are not required for the function of hY RNAs in chromosomal DNA replication in a human cell-free system, which can be mediated by Y RNAs outside of these RNPs. These data suggest that Y RNAs can support different cellular functions depending on associated proteins.     

Identification of a novel cis-acting RNA element involved in nuclear export of hY RNAs

Ro RNPs are small cytoplasmic RNA-protein complexes of unknown function that have been found in all metazoan cells studied so far. In human cells, Ro RNPs consist of one of four small RNA molecules, termed hY RNAs and at least two well-characterized proteins, Ro60 and La. In previous Xenopus laevis oocyte microinjection studies, we showed that an intact Ro60 binding site (Stem-loop 1) is a prerequisite for efficient nuclear export of hY1 RNA, whereas an intact La-binding site promotes nuclear retention (Simons et al. RNA, 1996, 2:264-273). Here we present evidence that the distal half (Stem 2) of the conserved base-paired stem structure found in all hY RNAs also plays a critical role in the export process. A minimal RNA molecule containing this region, L1S2 RNA, competes effectively for the export of full-length hY1 RNAs and is itself exported very rapidly in a Ro60-independent and RanGTP-dependent manner. Mutational analyses of this RNA shows that a 5'/3' terminal double-stranded stem structure (>10 bp) of no specific nucleotide sequence constitutes a novel nuclear export element (NEE). Cross-competition studies indicate that this type of NEE may also be involved in export of other classes of RNAs. Like full-length hY1 RNA, L1S2 RNA also competes for export of ET-202 RNA, an RNA that was selected for its efficient nuclear export in the presence of the nuclear transport inhibitor, VSV Matrix protein (Grimm et al. Proc Natl Acad Sci USA, 1997, 94:10122-10127). However, export of L1S2 RNA is strongly inhibited by VSV-M protein, showing that these RNAs use partially overlapping, but not identical export pathways. We propose that export of Y RNAs is mediated by two contiguous cis-acting elements in the 5'/3' double-stranded stem region that is conserved between different Y RNAs.     

Are the Ro RNP‐associated Y RNAs concealing microRNAs? Y RNA‐derived miRNAs may be involved in autoimmunity

Here we discuss the hypothesis that the RNA components of the Ro ribonucleoproteins (RNPs), the Y RNAs, can be processed into microRNAs (miRNAs). Although Ro RNPs, whose main protein components Ro60 and La are targeted by the immune system in several autoimmune diseases, were discovered many years ago, their function is still poorly understood. Indeed, recent data show that miRNA-sized small RNAs can be generated from Y RNAs. This hypothesis leads also to a model in which Ro60 acts as a modulator in the Y RNA-derived miRNA biogenesis pathway. The implications of these Y RNA-derived miRNAs, which may be specifically produced under pathological circumstances such as in autoimmunity or during viral infections, for the enigmatic function of Ro RNPs are discussed.     

Refined definition of the 56K and other autoantigens in the 50–60 kDa region

Alteration of the acrylamide: bisacrylamide ratio in the SDS-polyacrylamide gel used for Western blotting strongly improved the unambiguous detection of antibodies against 50–60 kDa autoantigens present in autoimmune patient sera. The relative migration of Ro 52, the 56K autoantigen and calreticulin increased with reduced acrylamide: bisacrylamide ratios in contrast to that of Ro60, La and Jo-1. These analyses indicated that these six autoantigens correspond to six distinct polypeptides.Further analyses using recombinant calreticulin showed that (i) the 56K autoantigen is neither identical nor related to calreticulin and (ii) calreticulin is not a Ro autoantigen.A series of experiments designed to better characterize the 56K autoantigen showed that (i) the antigen is not detectable in fixed cells, presumably due to masking of the epitopes; (ii) about equal amounts of the antigen were recovered in nuclear and cytoplasmic cell, fractions after enucleation of the cells; (iii) the 56K autoantigen is not stably associated with either RNA or other proteins.Abbreviations a- anti- - CaR calreticulin - NHS normal human serum - NRS normal rabbit serum - r recombinant - RA rheumatoid arthritis - SLE systemic lupus erythematosus - SS Sjögren's syndrome     

A subset of hY RNAs is associated with erythrocyte Ro ribonucleoproteins.

The Ro autoantigen is a mammalian cellular ribonucleoprotein (RNP) of unknown function. We have demonstrated that hY1 and hY4 Ro RNAs are associated with erythrocyte Ro RNPs and represent a subset of the four hY RNAs found in HeLa cell and leukocyte Ro RNPs. We have cloned and sequenced hY4 RNA, the only hY RNA not sequenced previously, from a polymerase chain reaction amplified erythrocyte hY cDNA library. Sequencing of the erythrocyte hY RNAs in conjunction with Northern blot analysis confirms that the erythrocyte hY RNAs contain the same sequences as the respective HeLa cell RNAs of similar mobility. Ribonuclease inhibition activity has been found in erythrocytes and this activity inhibits the degradation of hY3 and hY5 in leukocyte lysates thereby favoring the possibility that the presence of hY1 and hY4 in erythrocytes is the result of differential expression of the hY RNAs in erythrocyte precursors.     

狼疮La蛋白的分子生物学研究进展

狼疮La蛋白,又叫La核糖核蛋白、La自身抗原或干燥综合征B型抗原,是原发性干燥综合征的特异性自身抗原之一。La蛋白拥有多种结构和运输元件,可定位于不同的亚细胞部位与RNA相互作用。通常I丑蛋白主要存在于细胞核内,作为RNA聚合酶Ⅲ的转录因子,与RNA聚合酶Ⅲ转录新生产物结合,调节其转录终止,在转录后发挥分子伴侣作用,稳定RNA前体并促进其正确折叠,帮助其进行加工处理。La蛋白还可以与一类拥有内部核糖体进入位点的细胞内mRNA和病毒RNA结合,调节这类RNA的表达翻译;也可在细胞凋亡时被颗粒酶B酶解,产生特异性酶解片段,诱导特异性抗La自身抗体和干燥综合征的生成。我们就I丑蛋白的分子生物学方面的近期研究进展进行综述。     

Ro ribonucleoprotein assembly in vitro. Identification of RNA-protein and protein-protein interactions.

The human Y RNAs, small RNAs with an unknown function, are complexed with at least three proteins: the 60,000 M(r) Ro protein (Ro60), the 52,000 M(r) Ro protein (Ro52) and the La protein (La). In this study we examined the intermolecular interactions between the components of these so-called Ro ribonucleoprotein (Ro RNP) complexes. Incubation of 32P-labelled hY1 RNA in HeLa S100 extract allows the reconstitution of Ro RNP complexes, which were analysed by immunoprecipitation with monospecific antisera. By immunodepletion of HeLa S100 extracts for either Ro60, Ro52 or La, followed by supplementation with recombinant Ro60 or La, it was demonstrated that both Ro60 and La bind to hY1 RNA directly without being influenced by one of the other proteins. However, binding of Ro52 to hY1 RNA required the presence of Ro60, which strongly suggests that the association of Ro52 with Ro RNPs is mediated by protein-protein interactions between Ro60 and Ro52.     

Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells.

The ribonucleoprotein (RNP) particles containing the Epstein-Barr virus-associated small RNAs EBER1 and EBER2 were analyzed to determine their RNA secondary structures and sites of RNA-protein interaction. The secondary structures were probed with nucleases and by chemical modification with single-strand-specific reagents, and the sites of modification or cleavage were mapped by primer extension. These data were used to develop secondary structures for the two RNAs, and likely sites of close RNA-protein contact were identified by comparing modification patterns for naked RNA and RNA in RNP particles. In addition, sites of interaction between each Epstein-Barr virus-encoded RNA (EBER) and the La antigen were identified by analyzing RNA fragments resistant to digestion by RNase A or T1 after immunoprecipitation by an anti-La serum sample from a lupus patient. Our results confirm earlier findings that the La protein binds to the 3' terminus of each molecule. Possible functions for the EBER RNPs are discussed.     

RNA chaperone activity of protein components of human Ro RNPs

Ro ribonucleoprotein (RNP) complexes are composed of one molecule of a small noncoding cytoplasmic RNA, termed Y RNA, and the two proteins Ro60 and La. Additional proteins such as hnRNP I, hnRNP K, or nucleolin have recently been shown to be associated with subpopulations of Y RNAs. Ro RNPs appear to be localized in the cytoplasm of all higher eukaryotic cells but their functions have remained elusive. To shed light on possible functions of Ro RNPs, we tested protein components of these complexes for RNA chaperone properties employing two in vitro chaperone assays and additionally an in vivo chaperone assay. In these assays the splicing activity of a group I intron is measured. La showed pronounced RNA chaperone activity in the cis-splicing assay in vitro and also in vivo, whereas no activity was seen in the trans-splicing assay in vitro. Both hnRNP I and hnRNP K exhibited strong chaperone activity in the two in vitro assays, however, proved to be cytotoxic in the in vivo assay. No chaperone activity was observed for Ro60 in vitro and a moderate activity was detected in vivo. In vitro chaperone activities of La and hnRNP I were completely inhibited upon binding of Y RNA. Taken together, these data suggest that the Ro RNP components La, hnRNP K, and hnRNP I possess RNA chaperone activity, while Ro60-Y RNA complexes might function as transporters, bringing other Y RNA binding proteins to their specific targets.     

Characterization of the autoantigen La (SS-B) as a dsRNA unwinding enzyme.

During the analysis of the La (SS-B) autoantigen for catalytic activities an ATP-dependent double-stranded RNA unwinding activity was detected. Both native and recombinant La proteins from different species displayed this activity, which could be inhibited by monospecific anti-La antibodies. La protein was able to melt dsRNA substrates with either two 3'-overhangs or a single 3'- and a 5'-overhang. Double-stranded RNAs with two 5'-overhangs were not unwound, indicating that at least one 3'-overhang is required for unwinding. Sequence elements of the La protein that might be involved in dsRNA unwinding, such as an evolutionarily conserved putative ATP-binding motif and an element that is homologous to the double-stranded RNA binding protein kinase PKR, are discussed.