The Subcellular Distribution of an RNA Quality Control Protein, the Ro Autoantigen, Is Regulated by Noncoding Y RNA Binding

The Ro autoantigen is a ring-shaped RNA-binding protein that binds misfolded RNAs in nuclei and is proposed to function in quality control. In the cytoplasm, Ro binds noncoding RNAs, called Y RNAs, that inhibit access of Ro to other RNAs. Ro also assists survival of mammalian cells and at least one bacterium after UV irradiation. In mammals, Ro undergoes dramatic localization changes after UV irradiation, changing from mostly cytoplasmic to predominantly nuclear. Here, we report that a second role of Y RNAs is to regulate the subcellular distribution of Ro. A mutant Ro protein that does not bind Y RNAs accumulates in nuclei. Ro also localizes to nuclei when Y RNAs are depleted. By assaying chimeric proteins in which portions of mouse Ro were replaced with bacterial Ro sequences, we show that nuclear accumulation of Ro after irradiation requires sequences that overlap the Y RNA binding site. Ro also accumulates in nuclei after oxidative stress, and similar sequences are required. Together, these data reveal that Ro contains a signal for nuclear accumulation that is masked by a bound Y RNA and suggest that Y RNA binding may be modulated during cell stress.     

Ro60 and Y RNAs: structure,functions, and roles in autoimmunity

Ro60, also known as SS-A or TROVE2, is an evolutionarily conserved RNA-binding protein that is found in most animal cells, approximately 5% of sequenced prokaryotic genomes and some archaea. Ro60 is present in cells as both a free protein and as a component of a ribonucleoprotein complex, where its best-known partners are members of a class of noncoding RNAs called Y RNAs. Structural and biochemical analyses have revealed that Ro60 is a ring-shaped protein that binds Y RNAs on its outer surface. In addition to Y RNAs, Ro60 binds misfolded and aberrant noncoding RNAs in some animal cell nuclei. Although the fate of these defective Ro60-bound noncoding RNAs in animal cells is not well-defined, a bacterial Ro60 ortholog functions with 3′ to 5′ exoribonucleases to assist structured RNA degradation. Studies of Y RNAs have revealed that these RNAs regulate the subcellular localization of Ro60, tether Ro60 to effector proteins and regulate the access of other RNAs to its central cavity. As both mammalian cells and bacteria lacking Ro60 are sensitized to ultraviolet irradiation, Ro60 function may be important during exposure to some environmental stressors. Here we summarize the current knowledge regarding the functions of Ro60 and Y RNAs in animal cells and bacteria. Because the Ro60 RNP is a clinically important target of autoantibodies in patients with rheumatic diseases such as Sjogren’s syndrome, systemic lupus erythematosus, and neonatal lupus, we also discuss potential roles for Ro60 RNPs in the initiation and pathogenesis of systemic autoimmune rheumatic disease.     

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.     

The Ro autoantigen binds misfolded U2 small nuclear RNAs and assists mammalian cell survival after UV irradiation

The Ro 60 kDa autoantigen, an RNA binding protein, is a major target of the immune response in patients with systemic lupus erythematosus. As mice lacking Ro develop a lupus-like syndrome, Ro may be important for preventing autoimmunity. However, the cellular function of Ro, which binds small cytoplasmic RNAs of unknown function called Y RNAs, has been enigmatic. Ro has been proposed to function in 5S rRNA quality control based on experiments in Xenopus laevis oocytes, and a Ro ortholog enhances survival of the eubacterium Deinococcus radiodurans after ultraviolet irradiation. To test the general importance of these two observations for Ro function, we investigated the role of Ro in mammalian cells. We report that, in mouse embryonic stem (ES) cells, Ro binds variant spliceosomal U2 snRNAs. Expression of mouse U2 snRNAs in Xenopus oocytes reveals that binding occurs in nuclei and appears to involve recognition of misfolded RNA. Moreover, mouse ES cells lacking Ro exhibit decreased survival after ultraviolet irradiation. In irradiated cells, both Ro and a Y RNA accumulate in nuclei. We propose that Ro plays a general role in small RNA quality control and that this function is important for cell survival after ultraviolet irradiation.     

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.     

Conserved features of Y RNAs revealed by automated phylogenetic secondary structure analysis.

Y RNAs are small 'cytoplasmic' RNAs which are components of the Ro ribonucleoprotein (RNP) complex. The core of this complex, which is found in the cell nuclei of higher eukaryotes as well as the cytoplasm, is composed of a complex between the 60 kDa Ro protein and Y RNAs. Human cells contain four distinct Y RNAs (Y1, Y3, Y4 and Y5), while other eukaryotes contain a variable number of Y RNA homologues. When detected in a particular species, the Ro RNP has been present in every cell type within that particular organism. This characteristic, along with its high conservation among vertebrates, suggests an important function for Ro RNP in cellular metabolism; however, this function has not yet been definitively elucidated. In order to identify conserved features of Y RNA sequences and structures which may be directly involved in Ro RNP function, a phylogenetic comparative analysis of Y RNAs has been performed. Sequences of Y RNA homologues from five vertebrate species have been obtained and, together with previously published Y RNA sequences, used to predict Y RNA secondary structures. A novel RNA secondary structure comparison algorithm, the suboptimal RNA analysis program, has been developed and used in conjunction with available algorithms to find phylogenetically conserved secondary structure models for YI, Y3 and Y4 RNAs. Short, conserved sequences within the Y RNAs have been identified and are invariant among vertebrates, consistent with a direct role for Y RNAs in Ro function. A subset of these are located wholly or partially in looped regions in the Y3 and Y4 RNA predicted model structures, in accord with the possibility that these Y RNAs base pair with other cellular nucleic acids or are sites of interaction between the Ro RNP and other macromolecules.     

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.     

Nuclear export of the DEAD box An3 protein by CRM1 is coupled to An3 helicase activity

We have recently identified the Xenopus laevis An3 protein as a bona fide substrate for the nuclear export receptor CRM1 (Exportin 1). An3 binds directly to CRM1 with high affinity via a leucine-rich nuclear export signal located in the extreme N terminus. An3 is a member of the DEAD box family of RNA helicases, which unwind RNA duplexes. RNA unwinding is coupled to hydrolysis of nucleoside triphosphates by the helicase, and the ATPase activity of several helicases is greatly stimulated by various polynucleotides. Here we report that dATP hydrolysis by An3 is stimulated approximately 6-fold by total RNA from X. laevis oocytes, whereas poly(U) RNA fails to enhance hydrolysis, suggesting the existence of a specific RNA activator for An3. Kinetic analysis reveals that a mutation within the conserved DEAD box motif reduces the rate of dATP hydrolysis by approximately 6-fold. In accordance with this, the DEAD box mutant is unable to unwind double-stranded RNA. Microinjection of the An3 DEAD box mutant into X. laevis oocytes nuclei reveals a significantly lower export rate as compared with wild-type An3 protein. This is not because the mutant has lower affinity toward CRM1, nor is it due to altered RNA binding capacity. This suggests that nuclear export of An3 protein by CRM1 is coupled to An3 helicase activity.     

Structural insights into RNA quality control: the Ro autoantigen binds misfolded RNAs via its central cavity

The Ro 60 kDa autoantigen is a major target of the immune response in patients with systemic lupus erythematosus. In vertebrate cells, Ro binds misfolded small RNAs and likely functions in RNA quality control. In eukaryotes and bacteria, Ro also associates with small RNAs called Y RNAs. We present structures of unliganded Ro and Ro complexed with two RNAs at 1.95 and 2.2 A resolution, respectively. Ro consists of a von Willebrand factor A domain and a doughnut-shaped domain composed of HEAT repeats. In the complex, a fragment of Y RNA binds on the outer surface of the HEAT-repeat ring, and single-stranded RNA binds in the toroid hole. Mutagenesis supports a binding site for misfolded RNAs that encompasses both sites, with a single-stranded end inserted into the toroid cavity. Our experiments suggest that one role of Y RNAs may be to regulate access of other RNAs to Ro.     

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.     

Crystal structure of Rsr, an ortholog of the antigenic Ro protein, links conformational flexibility to RNA binding activity

Ro ribonucleoproteins are a class of antigenic ribonucleoproteins associated with rheumatic autoimmune diseases like systemic lupus erythematosus and Sj?grens syndrome in humans. Ro ribonucleoproteins are mostly composed of the 60-kDa Ro protein and small cytoplasmic RNAs, called Y RNAs, of unknown function. In eukaryotes, where Ro has been found to associate with damaged or mutant RNAs, it has been suggested that Ro may play a role in RNA quality control. In the radiation-resistant bacterium Deinococcus radiodurans and some eukaryotes, Ro has also been implicated in cell survival following UV damage. Here we present the first high resolution structure of a prokaryotic Ro ortholog, Rsr from D. radiodurans. The structure has been solved to 1.9 A resolution and shows distinct differences when compared with the eukaryotic apo- and RNA-bound Ro structures. Rsr is composed of two domains: a helical RNA binding domain and a mixed "von Willebrand factor A-like" domain containing a divalent metal binding site. Although the individual domains of Rsr are similar to the eukaryotic Ro, significantly large differences are seen at the interface of the two domains. Since this interface communicates with the conserved central cavity of Ro, which is implicated in RNA binding, changes at this interface could potentially influence RNA binding by Ro. Although the apo-Rsr protein is monomeric, Rsr binds Y RNA to form multimers of approximately 12 molecules of a 1:1 Rsr-Y RNA complex. Rsr binds D. radiodurans Y RNA with low nanomolar affinity, comparable with previously characterized eukaryotic Ro orthologs.     

Regulation of the export of RNA from the nucleus

Transport of macromolecules across the nuclear envelope is an essential activity in eukaryotic cells. RNA molecules within cells are found complexed with proteins and the bound proteins likely contain signals for RNA export. RNAs microinjected into Xenopus oocyte nuclei are readily exported, and their export can be competed by self RNA but not by RNAs of other classes. This indicates that the rate-limiting step in RNA export is the interaction of RNAs with class-specific proteins, at least when substrate RNAs are present at saturating levels. Export of host mRNAs is inhibited following infection by some animal viruses, while the export of viral RNAs occurs. The HIV-1 RNA-binding protein, Rev, mediates the export of intron-containing viral RNAs that would normally be retained in nuclei. This requires a nuclear export signal (NES) within Rev and an element within the RNA to which Rev binds. In yeast, heat shock causes accumulation of poly(A)(+)RNA within nuclei but heat-shock mRNAs are transcribed and exported efficiently. This requires elements within heat shock mRNA that probably interact with a cellular protein to facilitate RNA export. In these cases, the proteins that recognize critical sequences in the RNAs probably direct the RNAs to an RNA export pathway not generally used for mRNA export. This would circumvent the general retention of most poly(A)(+)mRNAs following heat shock in yeast and the need for complete splicing of viral mRNAs that travel through the normal mRNA export pathway.     

Overexpressed LEF-1 proteins display different nuclear localization patterns of beta-catenin in normal versus tumor cells

Beta-catenin not only plays a role in cadherin-dependent cell adhesion, but also interacts with T-cell factor (TCF)/lymphoid enhancer factor-1 (LEF-1) to affect gene expression. In this report, we describe the effects of exogenous LEF-1 and of treatment with leptomycin B (LMB), a specific inhibitor of CRM1-medicated nuclear export, on the nuclear localization and export of beta-catenin. Normal epithelial cells overexpressing LEF-1 accumulate nuclear beta-catenin in a LEF-1 concentration-dependent manner. Nuclear beta-catenin, once imported from the cytoplasm, is rapidly removed from the nucleus. Treatment with LMB results in dramatic retention of nuclear beta-catenin in normal epithelial cells transfected with LEF-1, and this effect is intensified by treatment of N-Acetyl-leucyl-leucyl-norleucinal together with LMB. Colon carcinoma cells containing an adenomatous polyposis coli mutation retain significant amounts of LEF-1 induced nuclear beta-catenin considerably after the time-point when beta-catenin disappears from the nuclei of LEF-1 transfected normal epithelial cells. beta-Catenin binds directly to CRM1, and overexpression of CRM1 reduces nuclear beta-catenin-mediated transactivation function.     

CRM1 controls the composition of nucleoplasmic pre-snoRNA complexes to licence them for nucleolar transport

Transport of C/D snoRNPs to nucleoli involves nuclear export factors. In particular, CRM1 binds nascent snoRNPs, but its precise role remains unknown. We show here that both CRM1 and nucleocytoplasmic trafficking are required to transport snoRNPs to nucleoli, but the snoRNPs do not transit through the cytoplasm. Instead, CRM1 controls the composition of nucleoplasmic pre-snoRNP complexes. We observed that Tgs1 long form (Tgs1 LF), the long isoform of the cap hypermethylase, contains a leucine-rich nuclear export signal, shuttles in a CRM1-dependent manner, and binds to the nucleolar localization signal (NoLS) of the core snoRNP protein Nop58. In vitro data indicate that CRM1 binds Tgs1 LF and promotes its dissociation from Nop58 NoLS, and immunoprecipitation experiments from cells indicate that the association of Tgs1 LF with snoRNPs increases upon CRM1 inhibition. Thus, CRM1 appears to promote nucleolar transport of snoRNPs by removing Tgs1 LF from the Nop58 NoLS. Microarray/IP data show that this occurs on most snoRNPs, from both C/D and H/ACA families, and on the telomerase RNA. Hence, CRM1 provides a general molecular link between nuclear events and nucleocytoplasmic trafficking.     

The C protein of wild-type measles virus has the ability to shuttle between the nucleus and the cytoplasm

Measles virus (MV) C protein is a small and basic non-structural protein, but its function is not well understood. We have found that a FLAG-tagged wild-type MV C protein expressed from cDNA was accumulated exclusively in the nucleus. To analyze the amino acid sequence important for the nuclear localization of C protein, a plasmid expressing C protein fused to the enhanced green fluorescent protein (EGFP) was generated. Mutation analysis revealed that (41)PPARKRRQ(48), belonging to the classical nuclear localization signal was important for nuclear localization. Analysis of the amino acid sequence of C protein revealed that it has a nuclear export signal (NES)-like sequence, (76)LEKAMTTLKL(85). Addition of the putative NES to the EGFP resulted in the translocation of EGFP to the cytoplasm. The Rev(1.4)-EGFP nuclear export assay showed that this putative NES has a CRM1-dependent NES activity. C-EGFP accumulated in HeLa nuclei could be translocated to NIH3T3 nuclei in heterokaryon assays. In MV-infected cells, C-EGFP was accumulated in the nuclei in early phase but in the cytoplasm in late phase. These results indicate that the putative NES is functional and that C protein has the ability to shuttle between the nucleus and the cytoplasm.