CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology.

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

Transfer RNA halves are found as nicked tRNAs in cells: evidence that nicked tRNAs regulate expression of an RNA repair operon.

Transfer RNA fragments are proposed to regulate numerous processes in eukaryotes, including translation inhibition, epigenetic inheritance, and cancer. In the bacterium Salmonella enterica serovar Typhimurium, 5' tRNA halves ending in 2',3' cyclic phosphate are proposed to bind the RtcR transcriptional activator, resulting in transcription of an RNA repair operon. However, since 5' and 3' tRNA halves can remain base paired after cleavage, the 5' tRNA halves could potentially bind RtcR as nicked tRNAs. Here we report that nicked tRNAs are ligands for RtcR. By isolating RNA from bacteria under conditions that preserve base pairing, we show that many tRNA halves are in the form of nicked tRNAs. Using a circularly permuted tRNA that mimics a nicked tRNA, we show that nicked tRNA ending in 2',3' cyclic phosphate is a better ligand for RtcR than the corresponding 5' tRNA half. In human cells, we show that some tRNA halves similarly remain base paired as nicked tRNAs following cleavage by anticodon nucleases. Our work supports a role for the RNA repair operon in repairing nicked tRNAs and has implications for the functions proposed for tRNA fragments in eukaryotes.

Support for a career in science.

I am so very honored to receive the Women in Cell Biology Sandra K. Masur Senior Leadership Award from the American Society for Cell Biology (ASCB), particularly because many of the previous awardees have served as mentors and sources of inspiration throughout my own career. I also thank the ASCB for always striving to be maximally inclusive, in terms of both the scientists it supports and its broad vision of what constitutes cell biology. As a graduate student I gave one of my first talks at an ASCB meeting, and I am proud to have been an ASCB member for almost 30 years. In this essay, I describe my own career to illustrate the support that I believe is needed to achieve a career in science.

The Autoantigen Repertoire and the Microbial RNP World.

Although autoimmunity and autoimmune disease (AID) are relatively common, the repertoire of autoantigens is paradoxically very limited. Highly enriched in this autoantigen repertoire are nucleic acids and their binding proteins, which together form large macromolecular structures. Most of these complexes are of ancient evolutionary origin, with homologs throughout multiple kingdoms of life. Why and if these nucleic acid-protein particles drive the development of autoimmunity remains unresolved. Recent advances in our understanding of the microbiome may provide clues about the origins of autoimmunity - and the particular puzzle of why the autoantigen repertoire is so particularly enriched in ribonucleoprotein particles (RNPs). We discuss the possibility that autoimmunity to some RNPs may arise from molecular mimicry to microbial orthologs.

An RNA Repair Operon Regulated by Damaged tRNAs.

Many bacteria contain an RNA repair operon, encoding the RtcB RNA ligase and the RtcA RNA cyclase, that is regulated by the RtcR transcriptional activator. Although RtcR contains a divergent version of the CARF (CRISPR-associated Rossman fold) oligonucleotide-binding regulatory domain, both the specific signal that regulates operon expression and the substrates of the encoded enzymes are unknown. We report that tRNA fragments activate operon expression. Using a genetic screen in Salmonella enterica serovar Typhimurium, we find that the operon is expressed in the presence of mutations that cause tRNA fragments to accumulate. RtcA, which converts RNA phosphate ends to 2', 3'-cyclic phosphate, is also required. Operon expression and tRNA fragment accumulation also occur upon DNA damage. The CARF domain binds 5' tRNA fragments ending in cyclic phosphate, and RtcR oligomerizes upon binding these ligands, a prerequisite for operon activation. Our studies reveal a signaling pathway involving broken tRNAs and implicate the operon in tRNA repair.

The Bacterial Ro60 Protein and Its Noncoding Y RNA Regulators.

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. In D. 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.

Noncoding Y RNAs regulate the levels, subcellular distribution and protein interactions of their Ro60 autoantigen partner.

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.

A guide to naming human non-coding RNA genes.

Research on non-coding RNA (ncRNA) is a rapidly expanding field. Providing an official gene symbol and name to ncRNA genes brings order to otherwise potential chaos as it allows unambiguous communication about each gene. The HUGO Gene Nomenclature Committee (HGNC, www.genenames.org) is the only group with the authority to approve symbols for human genes. The HGNC works with specialist advisors for different classes of ncRNA to ensure that ncRNA nomenclature is accurate and informative, where possible. Here, we review each major class of ncRNA that is currently annotated in the human genome and describe how each class is assigned a standardised nomenclature.

Cellular RNA surveillance in health and disease.

The numerous quality control pathways that target defective ribonucleic acids (RNAs) for degradation play key roles in shaping mammalian transcriptomes and preventing disease. These pathways monitor most steps in the biogenesis of both noncoding RNAs (ncRNAs) and protein-coding messenger RNAs (mRNAs), degrading ncRNAs that fail to form functional complexes with one or more proteins and eliminating mRNAs that encode abnormal, potentially toxic proteins. Mutations in components of diverse RNA surveillance pathways manifest as disease. Some mutations are characterized by increased interferon production, suggesting that a major role of these pathways is to prevent aberrant cellular RNAs from being recognized as "non-self." Other mutations are common in cancer, or result in developmental defects, revealing the importance of RNA surveillance to cell and organismal function.

The RNA exosome nuclease complex regulates human embryonic stem cell differentiation.

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.

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.

Structural Basis for tRNA Mimicry by a Bacterial Y RNA.

Noncoding Y RNAs are present in both animal cells and many bacteria. In all species examined, Y RNAs tether the Ro60 protein to an effector protein to perform various cellular functions. Recently, a new Y RNA subfamily was identified in bacteria. Bioinformatic analyses of these YrlA (Y RNA-like A) RNAs predict that the effector-binding domain resembles tRNA. We present the structure of this domain, the overall folding of which is strikingly similar to canonical tRNAs. The tertiary interactions that are responsible for stabilizing tRNA are present in YrlA, making it a close tRNA mimic. However, YrlA lacks a free CCA end and contains a kink in the stem corresponding to the anticodon stem. Since nucleotides in the D and T stems are conserved among YrlAs, they may be an interaction site for an unknown factor. Our experiments identify YrlA RNAs as a new class of tRNA mimics.

Bacterial Y RNAs: Gates, Tethers, and tRNA Mimics.

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.

Commensal orthologs of the human autoantigen Ro60 as triggers of autoimmunity in lupus.

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.

Noncoding RNA Surveillance: The Ends Justify the Means.

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.

Recruitment of 7SL RNA to assembling HIV-1 virus-like particles.

Retroviruses incorporate specific host cell RNAs into virions. In particular, the host noncoding 7SL RNA is highly abundant in all examined retroviruses compared with its cellular levels or relative to common mRNAs such as actin. Using live cell imaging techniques, we have determined that the 7SL RNA does not arrive with the HIV-1 RNA genome. Instead, it is recruited contemporaneously with assembly of the protein HIV-1 Gag at the plasma membrane. Further, we demonstrate that complexes of 7SL RNA and Gag can be immunoprecipitated from both cytosolic and plasma membrane fractions. This indicates that 7SL RNAs likely interact with Gag prior to high-order Gag multimerization at the plasma membrane. Thus, the interactions between Gag and the host RNA 7SL occur independent of the interactions between Gag and the host endosomal sorting complex required for transport (ESCRT) proteins, which are recruited temporarily at late stages of assembly. The interactions of 7SL and Gag are also independent of interactions of Gag and the HIV-1 genome which are seen on the plasma membrane prior to assembly of Gag.

Accumulation of Antigen-Driven Lymphoproliferations in Complement Receptor 2/CD21-/low B Cells From Patients With Sjögren's Syndrome.

OBJECTIVE: Patients with Sjögren's syndrome (SS) are prone to develop malignant lymphomas, and a correlation has been established between the lymphoproliferations occurring in these disorders and the presence in patients' blood of an unusual B cell population that down-regulates complement receptor 2/CD21. This study was undertaken to identify the B cell compartment from which these lymphoproliferations emerge and determine the mechanisms that promote clonal B cell expansion in patients with SS. METHODS: The reactivity of antibodies expressed by CD19+CD10-CD27-IgM+CD21-/low cells isolated from the blood of patients with SS was tested using a polymerase chain reaction-based approach that allows us to clone and express, in vitro, recombinant antibodies produced by single B cells. RESULTS: Clonal expansions were identified in CD21-/low B cells isolated from the peripheral blood of 3 patients with SS. These lymphoproliferations expressed B cell receptors (BCRs) that displayed somatic hypermutation lineage trees characteristic of a strong selection by antigens; one of these antigens was identified as a ribosomal self antigen. When the mutated BCR sequences expressed by the expanded CD21-/low B cell clones from patients with SS were reverted in vitro to their germline counterparts, one clone remained autoreactive. CONCLUSION: Clonal lymphoproliferations in patients with SS preferentially accumulate in the autoreactive CD21-/low B cell compartment often expanded in these subjects, and recognition of self antigens may drive the clonal B cell expansion while further refining BCR self-reactivity.

The Host RNAs in Retroviral Particles.

As they assemble, retroviruses encapsidate both their genomic RNAs and several types of host RNA. Whereas limited amounts of messenger RNA (mRNA) are detectable within virion populations, the predominant classes of encapsidated host RNAs do not encode proteins, but instead include endogenous retroelements and several classes of non-coding RNA (ncRNA), some of which are packaged in significant molar excess to the viral genome. Surprisingly, although the most abundant host RNAs in retroviruses are also abundant in cells, unusual forms of these RNAs are packaged preferentially, suggesting that these RNAs are recruited early in their biogenesis: before associating with their cognate protein partners, and/or from transient or rare RNA populations. These RNAs' packaging determinants differ from the viral genome's, and several of the abundantly packaged host ncRNAs serve cells as the scaffolds of ribonucleoprotein particles. Because virion assembly is equally efficient whether or not genomic RNA is available, yet RNA appears critical to the structural integrity of retroviral particles, it seems possible that the selectively encapsidated host ncRNAs might play roles in assembly. Indeed, some host ncRNAs appear to act during replication, as some transfer RNA (tRNA) species may contribute to nuclear import of human immunodeficiency virus 1 (HIV-1) reverse transcription complexes, and other tRNA interactions with the viral Gag protein aid correct trafficking to plasma membrane assembly sites. However, despite high conservation of packaging for certain host RNAs, replication roles for most of these selectively encapsidated RNAs-if any-have remained elusive.

Analysis of the human immunodeficiency virus-1 RNA packageome.

All retroviruses package cellular RNAs into virions. Studies of murine leukemia virus (MLV) revealed that the major host cell RNAs encapsidated by this simple retrovirus were LTR retrotransposons and noncoding RNAs (ncRNAs). Several classes of ncRNAs appeared to be packaged by MLV shortly after synthesis, as precursors to tRNAs, small nuclear RNAs, and small nucleolar RNAs were all enriched in virions. To determine the extent to which the human immunodeficiency virus (HIV-1) packages similar RNAs, we used high-throughput sequencing to characterize the RNAs within infectious HIV-1 virions produced in CEM-SS T lymphoblastoid cells. We report that the most abundant cellular RNAs in HIV-1 virions are 7SL RNA and transcripts from numerous divergent and truncated members of the long interspersed element (LINE) and short interspersed element (SINE) families of retrotransposons. We also detected precursors to several tRNAs and small nuclear RNAs as well as transcripts derived from the ribosomal DNA (rDNA) intergenic spacers. We show that packaging of a pre-tRNA requires the nuclear export receptor Exportin 5, indicating that HIV-1 recruits at least some newly made ncRNAs in the cytoplasm. Together, our work identifies the set of RNAs packaged by HIV-1 and reveals that early steps in HIV-1 assembly intersect with host cell ncRNA biogenesis pathways.