Although ATP-independent chaperones assist RNA folding, the mechanisms by which they function remain elusive. Here, we demonstrate how two RNA chaperones collaborate to unfold misfolded noncoding RNAs (ncRNAs). The ring-shaped Ro60 protein binds the ends of misfolded ncRNAs in its cavity, whereas La stabilizes nascent ncRNAs and assists their folding. Using cryo-electron microscopy to resolve the structure of a misfolded RNA complexed with Ro60 and La, we show that La cradles the Ro60 ribonucleoprotein (RNP), with its N-terminal domain binding the RNA 3' end after it passes through the Ro60 cavity, while its C-terminal domain destabilizes structures in the misfolded RNA body. Using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), we show that La and Ro60 function synergistically to unfold non-native structures. As the RNAs bound by Ro60 and La include both ncRNA precursors and ncRNAs with oligouridine tails, this RNA chaperone machine may function widely to recognize misfolded and otherwise aberrant ncRNAs and assist their unfolding.

Although most studies of the RNAs within extracellular vesicles (EVs) have focused on messenger RNA (mRNA) and microRNAs (miRNAs), recent analyses have revealed that transfer RNAs (tRNAs) and other noncoding RNAs (ncRNAs) are far more abundant. However, the extent to which EV ncRNAs resemble overall cellular RNAs and the benefits to host cells of packaging them into EVs remain unknown. Here, we purified EVs from the culture media of mouse and human cells and characterized their RNA components using high-throughput sequencing and Northern blotting. We report that EVs are enriched for numerous aberrant ncRNAs, including ncRNA fragments, ncRNAs that have failed to mature, and short structured introns. Many RNAs contain oligouridine tails, a modification that can promote degradation by DIS3L2, an exoribonuclease that degrades defective structured ncRNAs. Both the numbers of EVs released and the fractions of tailed RNAs in EVs increase on DIS3L2 depletion, indicating that EV packaging of aberrant ncRNAs occurs in competition with DIS3L2 decay. Multiple type I interferon-stimulated genes (ISGs) are upregulated on DIS3L2 depletion, and cells treated with a neutral sphingomyelinase inhibitor that reduces exosome biogenesis show moderate ISG upregulation, an effect that is enhanced in DIS3L2-depleted cells and accompanied by accumulation of aberrant ncRNAs in cellular RNA. Thus, DIS3L2 degradation and packaging of aberrant RNAs into vesicles may prevent these RNAs from activating innate immune sensors and triggering an interferon response.

Immune-mediated diseases are common in humans. The immune system is a complex host defense system that evolved to protect us from pathogens, but also plays an important role in homeostatic processes, removing dead or senescent cells, and participating in tumor surveillance. The human immune system has two arms: the older innate immune system and the newer adaptive immune system. Sensing of foreign RNA is critical to the innate immune system's ability to recognize pathogens, especially viral infections. However, RNA sensors are also strongly implicated in autoimmune and autoinflammatory diseases, highlighting the importance of balancing pathogen recognition with tolerance to host RNAs that can resemble their viral counterparts. We describe how RNA sensors bind their ligands, how this binding is coupled to upregulation of type I interferon-stimulated genes, and the ways in which mutations in RNA sensors and genes that play important roles in RNA homeostasis have been linked to autoimmune and autoinflammatory diseases.

Noncoding Y RNAs are abundant in animal cells and present in many bacteria. These RNAs are bound and stabilized by Ro60, a ring-shaped protein that is a target of autoantibodies in patients with systemic lupus erythematosus. Studies in bacteria revealed that Y RNA tethers Ro60 to a ring-shaped exoribonuclease, forming a double-ringed RNP machine specialized for structured RNA degradation. In addition to functioning as a tether, the bacterial RNA gates access of substrates to the Ro60 cavity. To identify roles for Y RNAs in mammals, we used CRISPR to generate mouse embryonic stem cells lacking one or both of the two murine Y RNAs. Despite reports that animal cell Y RNAs are essential for DNA replication, cells lacking these RNAs divide normally. However, Ro60 levels are reduced, revealing that Y RNA binding is required for Ro60 to accumulate to wild-type levels. Y RNAs regulate the subcellular location of Ro60, since Ro60 is reduced in the cytoplasm and increased in nucleoli when Y RNAs are absent. Last, we show that Y RNAs tether Ro60 to diverse effector proteins to generate specialized RNPs. Together, our data demonstrate that the roles of Y RNAs are intimately connected to that of their Ro60 partner.

Ro60 ribonucleoproteins (RNPs), composed of the ring-shaped Ro 60-kDa (Ro60) protein and noncoding RNAs called Y RNAs, are present in all three domains of life. Ro60 was first described as an autoantigen in patients with rheumatic disease, and Ro60 orthologs have been identified in 3% to 5% of bacterial genomes, spanning the majority of phyla. Their functions have been characterized primarily in Deinococcus radiodurans, the first sequenced bacterium with a recognizable ortholog. InD. radiodurans, the Ro60 ortholog enhances the ability of 3'-to-5' exoribonucleases to degrade structured RNA during several forms of environmental stress. Y RNAs are regulators that inhibit or allow the interactions of Ro60 with other proteins and RNAs. Studies of Ro60 RNPs in other bacteria hint at additional functions, since the most conserved Y RNA contains a domain that is a close tRNA mimic and Ro60 RNPs are often encoded adjacent to components of RNA repair systems.

A defining feature of embryonic stem cells (ESCs) is the ability to differentiate into all three germ layers. Pluripotency is maintained in part by a unique transcription network that maintains expression of pluripotency-specific transcription factors and represses developmental genes. While the mechanisms that establish this transcription network are well studied, little is known of the posttranscriptional surveillance pathways that degrade differentiation-related RNAs. We report that the surveillance pathway mediated by the RNA exosome nuclease complex represses ESC differentiation. Depletion of the exosome expedites differentiation of human ESCs into all three germ layers. LINE-1 retrotransposons and specific miRNAs, lncRNAs, and mRNAs that encode developmental regulators or affect their expression are all bound by the exosome and increase in level upon exosome depletion. The exosome restrains differentiation in part by degrading transcripts encoding FOXH1, a transcription factor crucial for mesendoderm formation. Our studies establish the exosome as a regulator of human ESC differentiation and reveal the importance of RNA decay in maintaining pluripotency.

The earliest autoantibodies in lupus are directed against the RNA binding autoantigen Ro60, but the triggers against this evolutionarily conserved antigen remain elusive. We identified Ro60 orthologs in a subset of human skin, oral, and gut commensal bacterial species and confirmed the presence of these orthologs in patients with lupus and healthy controls. Thus, we hypothesized that commensal Ro60 orthologs may trigger autoimmunity via cross-reactivity in genetically susceptible individuals. Sera from human anti-Ro60-positive lupus patients immunoprecipitated commensal Ro60 ribonucleoproteins. Human Ro60 autoantigen-specific CD4 memory T cell clones from lupus patients were activated by skin and mucosal Ro60-containing bacteria, supporting T cell cross-reactivity in humans. Further, germ-free mice spontaneously initiated anti-human Ro60 T and B cell responses and developed glomerular immune complex deposits after monocolonization with a Ro60 ortholog-containing gut commensal, linking anti-Ro60 commensal responses in vivo with the production of human Ro60 autoantibodies and signs of autoimmunity. Together, these data support that colonization with autoantigen ortholog-producing commensal species may initiate and sustain chronic autoimmunity in genetically predisposed individuals. The concept of commensal ortholog cross-reactivity may apply more broadly to autoimmune diseases and lead to novel treatment approaches aimed at defined commensal species.

Y RNAs are noncoding RNAs (ncRNAs) that are present in most animal cells and also in many bacteria. These RNAs were discovered because they are bound by the Ro60 protein, a major target of autoantibodies in patients with some systemic autoimmune rheumatic diseases. Studies of Ro60 and Y RNAs in Deinococcus radiodurans, the first sequenced bacterium with a Ro60 ortholog, revealed that they function with 3'-to-5' exoribonucleases to alter the composition of RNA populations during some forms of environmental stress. In the best-characterized example, Y RNA tethers the Ro60 protein to the exoribonuclease polynucleotide phosphorylase, allowing this exoribonuclease to degrade structured RNAs more effectively. Y RNAs can also function as gates to regulate access of other RNAs to the Ro60 central cavity. Recent studies in the enteric bacterium Salmonella enterica serovar Typhimurium resulted in the discovery that Y RNAs are widely present in bacteria. Remarkably, the most-conserved subclass of bacterial Y RNAs contains a domain that mimics tRNA. In this review, we discuss the structure, conservation, and known functions of bacterial Y RNAs as well as the certainty that more bacterial Y RNAs and additional roles for these ncRNAs remain to be uncovered.

Anti-Ro60 autoantibodies are some of the earliest found in lupus patients and initiate epitope spreading. We identified a subset of commensal microbes with Ro60 orthologs, and hypothesized that Ro60 cross-reactivity may initiate or flare lupus in genetically susceptible individuals. Subjects with systemic and subacute cutaneous lupus erythematosus and healthy controls were recruited and 16S V4 sequencing of the skin, oral, and fecal microbiomes was performed. The presence of commensals with Ro60 orthologs was common among healthy and lupus subjects. Ro60+ lupus subjects had higher mean levels of P. propionicum on the skin than healthy subjects but there was no overall dysbiosis of the microbiota. Lupus memory T cell clones specific for P. propionicum proliferated in response to human Ro60. Similarly, T cell clones specific for B. thetaiotaomicron Ro60 proliferated in response to human Ro60, demonstrating T cell cross-reactivity. Human Ro60+ lupus serum immunoprecipitated P. propionicum Ro60 and its Y RNA binding partner, suggesting B cell cross-reactivity. Mice monocolonized with B. thetaiotaomicron produced serum anti-Ro60 antibodies, and cells from spleen and mesenteric lymph node proliferated in response to both B. thetaiotaomicron Ro60 and human Ro60, demonstrating cross-reactivity and the potential for causality. In summary, Ro60 autoimmune T and B cells from human lupus patients reacted with commensal Ro60 in vitro, and commensal Ro60 triggered anti-human Ro60 responses in vivo. Taken together, these data support that colonization with Ro60 ortholog-producing bacteria may induce and sustain chronic autoimmunity in lupus. This concept may apply more broadly to human autoimmune diseases and could lead to development of novel microbiota-targeted approaches to treat autoimmunity.

BackgroundThe earliest autoantibodies in systemic lupus erythematosus (SLE), a prototypical autoimmune disease, are directed against the RNA binding protein Ro60 but initiating triggers remain unknown. We identified commensal Ro60 orthologs in human skin, oral, and gut microbiomes in a subset of human species. Thus we hypothesize that Ro60‐ortholog‐carrying commensals induce autoimmunity via cross‐reactivity with human Ro60 in genetically susceptible individuals.MethodsV4 16S rDNA samples from SLE and control subjects were sequenced using the MiSeq platform. In addition, species‐specific enrichment by real‐time PCR for Ro60 bacteria (Propionibacterium propionicum, Corynebacterium amycolatum and Bacteroides thetaiotaomicron) was performed. Co‐immunoprecipitation was performed using human SLE serum and Ro60 ortholog‐containing bacterial lysates. Ro60‐specific memory CD4 T cells from SLE patients were cloned using a T cell library assay and stimulated with heat‐killed Ro60 bacteria. Supernatants were screened for cytokine proliferation using Legendplex. Ro60−/− mice were crossed to the TLR7.1 C57BL/6 transgenic lupus model and screened for Ro60 antibodies. Germ‐free mice were monocolonized with B. thetaiotaomicron and tested for anti‐Ro60 antibodies by ELISA. Mesenteric lymph node (MLN) and spleen cells were stimulated with bacterial and human Ro60 to assess for proliferation in vitro.ResultsRo60‐producing gut commensals were prevalent in controls and lupus patients. However, when human serum was used to co‐immunoprecipitate Ro60 and its bound Y RNA from the Ro60+ skin commensal P. propionicum, only antibodies from human Ro60‐positive lupus patients reacted with commensal Ro60. Lack of binding in Ro60‐negative patients or healthy controls supported antibody cross‐reactivity between human and commensal Ro60. Further, Human Ro60‐specific CD4 memory T cell clones proliferated in response to P. propionicum and commensal‐specific peptides, demonstrating T cell cross‐reactivity with commensal Ro60. Cytokine analysis proposes a heterogenous phenotype that is consistent with pathogen‐specific T cell clones. Serum anti‐Ro60 IgG autoantibodies were persistently induced in Ro60−/− TLR7 tg mice, suggesting a microbial trigger in the absence of host Ro60. Finally, MLN and splenic lymphocytes from germ‐free C57BL/6 mice that were monocolonized with B. thetaiotaomicron proliferated in response to bacterial and human Ro60 and sera contained anti‐human Ro60 IgG antibodies.ConclusionsAnti‐human Ro60 IgG in Ro60−/− TLR7 tg mice suggests a microbial trigger of these autoantibodies. Monocolonization with Ro60+ bacteria induced cross‐reactive anti‐human Ro60 responses in vivo. Ortholog cross‐reactivity is underscored by Ro60‐specific T cell clones and sera from lupus patients that reacted with commensal Ro60 in vitro. Our data support a model in which colonization with Ro60 ortholog‐carrying skin and gut bacteria sustain chronic autoreactivity in lupus. Quantifying and targeting Ro60+ bacteria in SLE patients may lead to novel biomarkers and treatment approaches.Ortholog cross‐reactivity is a novel concept that could contribute to the pathogenesis of human autoimmune diseases more broadly.Support or Funding InformationArthritis Foundation

Numerous surveillance pathways sculpt eukaryotic transcriptomes by degrading unneeded, defective, and potentially harmful noncoding RNAs (ncRNAs). Because aberrant and excess ncRNAs are largely degraded by exoribonucleases, a key characteristic of these RNAs is an accessible, protein-free 5' or 3' end. Most exoribonucleases function with cofactors that recognize ncRNAs with accessible 5' or 3' ends and/or increase the availability of these ends. Noncoding RNA surveillance pathways were first described in budding yeast, and there are now high-resolution structures of many components of the yeast pathways and significant mechanistic understanding as to how they function. Studies in human cells are revealing the ways in which these pathways both resemble and differ from their yeast counterparts, and are also uncovering numerous pathways that lack equivalents in budding yeast. In this review, we describe both the well-studied pathways uncovered in yeast and the new concepts that are emerging from studies in mammalian cells. We also discuss the ways in which surveillance pathways compete with chaperone proteins that transiently protect nascent ncRNA ends from exoribonucleases, with partner proteins that sequester these ends within RNPs, and with end modification pathways that protect the ends of some ncRNAs from nucleases.

Although all retroviruses recruit host cell RNAs into virions, both the spectrum of RNAs encapsidated and the mechanisms by which they are recruited remain largely unknown. Here, we used high-throughput sequencing to obtain a comprehensive description of the RNAs packaged by a model retrovirus, murine leukemia virus. The major encapsidated host RNAs are noncoding RNAs (ncRNAs) and members of the VL30 class of endogenous retroviruses. Remarkably, although Moloney leukemia virus (MLV) assembles in the cytoplasm, precursors to specific tRNAs, small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) are all enriched in virions. Consistent with their cytoplasmic recruitment, packaging of both pre-tRNAs and U6 snRNA requires the nuclear export receptor Exportin-5. Adenylated and uridylated forms of these RNAs accumulate in cells and virions when the cytoplasmic exoribonuclease DIS3L2 and subunits of the RNA exosome are depleted. Together, our data reveal that MLV recruits RNAs from a novel host cell surveillance pathway in which unprocessed and unneeded nuclear ncRNAs are exported to the cytoplasm for degradation.

Many bacteria encode an ortholog of the Ro60 autoantigen, a ring-shaped protein that is bound in animal cells to noncoding RNAs (ncRNAs) called Y RNAs. Studies in Deinococcus radiodurans revealed that Y RNA tethers Ro60 to polynucleotide phosphorylase, specializing this exoribonuclease for structured RNA degradation. Although Ro60 orthologs are present in a wide range of bacteria, Y RNAs have been detected in only two species, making it unclear whether these ncRNAs are common Ro60 partners in bacteria. In this study, we report that likely Y RNAs are encoded near Ro60 in >250 bacterial and phage species. By comparing conserved features, we discovered that at least one Y RNA in each species contains a domain resembling tRNA. We show that these RNAs contain nucleotide modifications characteristic of tRNA and are substrates for several enzymes that recognize tRNAs. Our studies confirm the importance of Y RNAs in bacterial physiology and identify a new class of ncRNAs that mimic tRNA.

Non-coding RNAs (ncRNAs) called Y RNAs are abundant components of both animal cells and a variety of bacteria. In all species examined, these ~100 nt RNAs are bound to the Ro 60 kDa (Ro60) autoantigen, a ring-shaped protein that also binds misfolded ncRNAs in some vertebrate nuclei. Although the function of Ro60 RNPs has been mysterious, we recently reported that a bacterial Y RNA tethers Ro60 to the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase) to form RYPER (Ro60/Y RNA/PNPase Exoribonuclease RNP), a new RNA degradation machine. PNPase is a homotrimeric ring that degrades single-stranded RNA, and Y RNA-mediated tethering of Ro60 increases the effectiveness of PNPase in degrading structured RNAs. Single particle electron microscopy of RYPER suggests that RNA threads through the Ro60 ring into the PNPase cavity. Further studies indicate that Y RNAs may also act as gates to regulate entry of RNA substrates into the Ro60 channel. These findings reveal novel functions for Y RNAs and raise questions about how the bacterial findings relate to the roles of these ncRNAs in animal cells. Here we review the literature on Y RNAs, highlighting their close relationship with Ro60 proteins and the hypothesis that these ncRNAs function generally to tether Ro60 rings to diverse RNA-binding proteins.

Cardiac neonatal lupus (NL) is presumed to arise from maternal autoantibody targeting an intracellular ribonucleoprotein, Ro60, which binds noncoding Y RNA and only becomes accessible to autoantibodies during apoptosis. Despite the importance of Ro60 trafficking in the development of cardiac NL, the mechanism underlying cell surface exposure is unknown. To evaluate the influence of Y RNA on the subcellular location of Ro60 during apoptosis and activation of macrophages, stable Ro60 knockout murine fibroblasts expressing wild-type or mutated FLAG-Ro60 were assessed. FLAG3-Ro60(K170A R174A) binds Y RNA, whereas FLAG3-Ro60(H187S) does not bind Y RNA; fibroblasts expressing these constructs showed equivalent intracellular expression of Ro60. In contrast, apoptotic fibroblasts containing FLAG3-Ro60(K170A R174A) were bound by anti-Ro60, whereas FLAG3-Ro60(H187S) was not surface expressed. RNA interference of mY3 RNA in wild-type fibroblasts inhibited surface translocation of Ro60 during apoptosis, whereas depletion of mY1 RNA did not affect Ro60 exposure. Furthermore, Ro60 was not exposed following overexpression of mY1 in the mY3-depleted fibroblasts. In an in vitro model of anti-Ro60–mediated injury, Y RNA was shown to be an obligate factor for TLR-dependent activation of macrophages challenged with anti-Ro60–opsonized apoptotic fibroblasts. Murine Y3 RNA is a necessary factor to support the surface translocation of Ro60, which is pivotal to the formation of immune complexes on apoptotic cells and a TLR-dependent proinflammatory cascade. Accordingly, the Y3 RNA moiety of the Ro60 ribonucleoprotein imparts a critical role in the pathogenicity of maternal anti-Ro60 autoantibodies.

Non-coding RNAs (ncRNAs) called Y RNAs are abundant components of both animal cells and a variety of bacteria. In all species examined, these ~100 nt RNAs are bound to the Ro 60 kDa (Ro60) autoantigen, a ring-shaped protein that also binds misfolded ncRNAs in some vertebrate nuclei. Although the function of Ro60 RNPs has been mysterious, we recently reported that a bacterial Y RNA tethers Ro60 to the 3' to 5' exoribonuclease polynucleotide phosphorylase (PNPase) to form RYPER (Ro60/Y RNA/PNPase Exoribonuclease RNP), a new RNA degradation machine. PNPase is a homotrimeric ring that degrades single-stranded RNA, and Y RNA-mediated tethering of Ro60 increases the effectiveness of PNPase in degrading structured RNAs. Single particle electron microscopy of RYPER suggests that RNA threads through the Ro60 ring into the PNPase cavity. Further studies indicate that Y RNAs may also act as gates to regulate entry of RNA substrates into the Ro60 channel. These findings reveal novel functions for Y RNAs and raise questions about how the bacterial findings relate to the roles of these ncRNAs in animal cells. Here we review the literature on Y RNAs, highlighting their close relationship with Ro60 proteins and the hypothesis that these ncRNAs function generally to tether Ro60 rings to diverse RNA-binding proteins.

Nuclear noncoding RNA (ncRNA) surveillance pathways play key roles in shaping the steady-state transcriptomes of eukaryotic cells. Defective and unneeded ncRNAs are primarily degraded by exoribonucleases that rely on protein cofactors to identify these RNAs. Recent studies have begun to elucidate both the mechanisms by which these cofactors recognize aberrant RNAs and the features that mark RNAs for degradation. One crucial RNA determinant is the presence of an accessible end; in addition, the failure of aberrant RNAs to fold into compact structures and assemble with specific binding proteins probably also contributes to their recognition and subsequent degradation. To date, ncRNA surveillance has been most extensively studied in budding yeast. However, mammalian cells possess nucleases and cofactors that have no known yeast counterparts, indicating that RNA surveillance pathways may be more complex in metazoans. Importantly, there is evidence that the failure of ncRNA surveillance pathways contributes to human disease.