RESUMEN
Detection of viruses by innate immune sensors induces protective antiviral immunity. The viral DNA sensor cyclic GMP-AMP synthase (cGAS) is necessary for detection of HIV by human dendritic cells and macrophages. However, synthesis of HIV DNA during infection is not sufficient for immune activation. The capsid protein, which associates with viral DNA, has a pivotal role in enabling cGAS-mediated immune activation. We now find that NONO is an essential sensor of the HIV capsid in the nucleus. NONO protein directly binds capsid with higher affinity for weakly pathogenic HIV-2 than highly pathogenic HIV-1. Upon infection, NONO is essential for cGAS activation by HIV and cGAS association with HIV DNA in the nucleus. NONO recognizes a conserved region in HIV capsid with limited tolerance for escape mutations. Detection of nuclear viral capsid by NONO to promote DNA sensing by cGAS reveals an innate strategy to achieve distinction of viruses from self in the nucleus.
Asunto(s)
Proteínas de la Cápside/inmunología , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas Asociadas a Matriz Nuclear/fisiología , Factores de Transcripción de Octámeros/inmunología , Factores de Transcripción de Octámeros/fisiología , Proteínas de Unión al ARN/inmunología , Proteínas de Unión al ARN/fisiología , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/fisiología , Núcleo Celular/metabolismo , ADN Viral/genética , ADN Viral/inmunología , Proteínas de Unión al ADN , Células Dendríticas/inmunología , Infecciones por VIH/inmunología , VIH-1/genética , VIH-1/inmunología , VIH-2/genética , VIH-2/inmunología , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata/inmunología , Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/fisiología , Proteínas de Unión al ARN/metabolismo , Transducción de Señal/inmunologíaRESUMEN
A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.
Asunto(s)
ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Secuencia de Bases , Sistemas CRISPR-Cas , Núcleo Celular/metabolismo , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Estabilidad del ARN , Factores de Transcripción/metabolismoRESUMEN
High-risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA-binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO-dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome-wide molecular analysis to reveal complex NONO-dependent regulation of gene expression. NONO forms RNA- and DNA-tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5' end of pre-mRNAs and modulates pre-mRNA processing, dependent on its RNA-binding activity. NONO regulates super-enhancer-associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA-binding activity of NONO may have therapeutic potential in this cancer context.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas de Unión al ADN , Neuroblastoma , Humanos , ADN/metabolismo , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA2/genética , Factor de Transcripción GATA2/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Factores de Transcripción/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
Delving deeply into the locus deleted in Prader-Willi syndrome, in this issue of Molecular Cell, Wu et al. (2016) identify SPA RNAs, a new class of 5' snoRNA-capped lncRNAs that sequester RNA binding proteins and influence alternative splicing.
Asunto(s)
Secuencia de Bases , Síndrome de Prader-Willi/genética , ARN Largo no Codificante/genética , ARN Nucleolar Pequeño/genética , Eliminación de Secuencia , Transcripción Genética , Empalme Alternativo , Cromosomas Humanos Par 15 , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Sitios Genéticos , Impresión Genómica , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Síndrome de Prader-Willi/metabolismo , Síndrome de Prader-Willi/patología , Unión Proteica , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Largo no Codificante/metabolismo , ARN Nucleolar Pequeño/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The Drosophila behaviour/human splicing (DBHS) proteins are a family of RNA/DNA binding cofactors liable for a range of cellular processes. DBHS proteins include the non-POU domain-containing octamer-binding protein (NONO) and paraspeckle protein component 1 (PSPC1), proteins capable of forming combinatorial dimers. Here, we describe the crystal structures of the human NONO and PSPC1 homodimers, representing uncharacterized DBHS dimerization states. The structures reveal a set of conserved contacts and structural plasticity within the dimerization interface that provide a rationale for dimer selectivity between DBHS paralogues. In addition, solution X-ray scattering and accompanying biochemical experiments describe a mechanism of cooperative RNA recognition by the NONO homodimer. Nucleic acid binding is reliant on RRM1, and appears to be affected by the orientation of RRM1, influenced by a newly identified 'ß-clasp' structure. Our structures shed light on the molecular determinants for DBHS homo- and heterodimerization and provide a basis for understanding how DBHS proteins cooperatively recognize a broad spectrum of RNA targets.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Dimerización , Humanos , Modelos Moleculares , Conformación Proteica , Empalme del ARNRESUMEN
RNA-binding proteins of the DBHS (Drosophila Behavior Human Splicing) family, NONO, SFPQ, and PSPC1 have numerous roles in genome stability and transcriptional and posttranscriptional regulation. Critical to DBHS activity is their recruitment to distinct subnuclear locations, for example, paraspeckle condensates, where DBHS proteins bind to the long noncoding RNA NEAT1 in the first essential step in paraspeckle formation. To carry out their diverse roles, DBHS proteins form homodimers and heterodimers, but how this dimerization influences DBHS localization and function is unknown. Here, we present an inducible GFP-NONO stable cell line and use it for live-cell 3D-structured illumination microscopy, revealing paraspeckles with dynamic, twisted elongated structures. Using siRNA knockdowns, we show these labeled paraspeckles consist of GFP-NONO/endogenous SFPQ dimers and that GFP-NONO localization to paraspeckles depends on endogenous SFPQ. Using purified proteins, we confirm that partner swapping between NONO and SFPQ occurs readily in vitro. Crystallographic analysis of the NONO-SFPQ heterodimer reveals conformational differences to the other DBHS dimer structures, which may contribute to partner preference, RNA specificity, and subnuclear localization. Thus overall, our study suggests heterodimer partner availability is crucial for NONO subnuclear distribution and helps explain the complexity of both DBHS protein and paraspeckle dynamics through imaging and structural approaches.
Asunto(s)
Paraspeckles , ARN Largo no Codificante , Humanos , Dimerización , Proteínas de Unión al ARN/metabolismo , Regulación de la Expresión Génica , ARN Largo no Codificante/genéticaRESUMEN
Paraspeckles are nuclear condensates, or membranelees organelles, that are built on the long noncoding RNA, NEAT1, and have been linked to many diseases. Although originally described as constitutive structures, here, in reviewing this field, we develop the hypothesis that cells increase paraspeckle abundance as part of a general stress response, to aid pro-survival pathways. Paraspeckles increase in many scenarios: when cells transform from one state to another, become infected with viruses and bacteria, begin to degenerate, under inflammation, in aging, and in cancer. Cells increase paraspeckles by increasing transcription of NEAT1 and adjusting its RNA processing. These increases in NEAT1 are driven by numerous stress-sensing signaling pathways, including signaling to mitochondria and stress granules, revealing crosstalk between the cytoplasm and nucleoplasm in the stress response. Thus, paraspeckles are an important piece of the puzzle in cellular homeostasis, and could be considered RNA-scaffolded nuclear equivalents of dynamic stress-induced structures that form in the cytoplasm. We speculate that, in general, cells rely on phase-separated paraspeckles to transiently tweak gene regulation in times of cellular flux.
Asunto(s)
Núcleo Celular , ARN Largo no Codificante , Regulación de la Expresión Génica , ARN Largo no Codificante/genéticaRESUMEN
Long noncoding RNA (lncRNA) molecules are some of the newest and least understood players in gene regulation. Hence, we need good model systems with well-defined RNA and protein components. One such system is paraspeckles - protein-rich nuclear organelles built around a specific lncRNA scaffold. New discoveries show how paraspeckles are formed through multiple RNA-protein and protein-protein interactions, some of which involve extensive polymerization, and others with multivalent interactions driving phase separation. Once formed, paraspeckles influence gene regulation through sequestration of component proteins and RNAs, with subsequent depletion in other compartments. Here we focus on the dual aspects of paraspeckle structure and function, revealing an emerging role for these dynamic bodies in a multitude of cellular settings.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , Animales , Humanos , Transición de Fase , ARN Largo no Codificante/genéticaRESUMEN
Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed paraspeckles, whereas NEAT1_1 is involved in paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neuroblastoma/genética , Oligonucleótidos Antisentido/genética , Poli A/genética , ARN Largo no Codificante/genética , Línea Celular Tumoral , Estudios de Cohortes , Perfilación de la Expresión Génica/métodos , Humanos , Estimación de Kaplan-Meier , Neuroblastoma/metabolismo , Neuroblastoma/patología , Poli A/metabolismo , Isoformas de ARN/genética , Isoformas de ARN/metabolismoRESUMEN
Members of the Drosophila behavior human splicing (DBHS) protein family are nuclear proteins implicated in many layers of nuclear functions, including RNA biogenesis as well as DNA repair. Definitive of the DBHS protein family, the conserved DBHS domain provides a dimerization platform that is critical for the structural integrity and function of these proteins. The three human DBHS proteins, splicing factor proline- and glutamine-rich (SFPQ), paraspeckle component 1 (PSPC1), and non-POU domain-containing octamer-binding protein (NONO), form either homo- or heterodimers; however, the relative affinity and mechanistic details of preferential heterodimerization are yet to be deciphered. Here we report the crystal structure of a SFPQ/PSPC1 heterodimer to 2.3-Å resolution and analyzed the subtle structural differences between the SFPQ/PSPC1 heterodimer and the previously characterized SFPQ homodimer. Analytical ultracentrifugation to estimate the dimerization equilibrium of the SFPQ-containing dimers revealed that the SFPQ-containing dimers dissociate at low micromolar concentrations and that the heterodimers have higher affinities than the homodimer. Moreover, we observed that the apparent dissociation constant for the SFPQ/PSPC1 heterodimer was over 6-fold lower than that of the SFPQ/NONO heterodimer. We propose that these differences in dimerization affinity may represent a potential mechanism by which PSPC1 at a lower relative cellular abundance can outcompete NONO to heterodimerize with SFPQ.
Asunto(s)
Proteínas Nucleares/química , Factor de Empalme Asociado a PTB/química , Multimerización de Proteína , Proteínas de Unión al ARN/química , Cristalografía por Rayos X , Humanos , Proteínas Nucleares/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , Estructura Cuaternaria de Proteína , Proteínas de Unión al ARN/metabolismoRESUMEN
De novo and inherited mutations of X-chromosome cell adhesion molecule protocadherin 19 (PCDH19) cause frequent, highly variable epilepsy, autism, cognitive decline and behavioural problems syndrome. Intriguingly, hemizygous null males are not affected while heterozygous females are, contradicting established X-chromosome inheritance. The disease mechanism is not known. Cellular mosaicism is the likely driver. We have identified p54nrb/NONO, a multifunctional nuclear paraspeckle protein with known roles in nuclear hormone receptor gene regulation, as a PCDH19 protein interacting partner. Using breast cancer cells we show that PCDH19-NONO complex is a positive co-regulator of ERα-mediated gene expression. Expression of mutant PCDH19 affects at least a subset of known ERα-regulated genes. These data are consistent with our findings that genes regulated by nuclear hormone receptors and those involved in the metabolism of neurosteroids in particular are dysregulated in PCDH19-epilepsy girls and affected mosaic males. Overall we define and characterize a novel mechanism of gene regulation driven by PCDH19, which is mediated by paraspeckle constituent NONO and is ERα-dependent. This PCDH19-NONO-ERα axis is of relevance not only to PCDH19-epilepsy and its comorbidities but likely also to ERα and generally nuclear hormone receptor-associated cancers.
Asunto(s)
Cadherinas/metabolismo , Proteínas Asociadas a Matriz Nuclear/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Proteínas de Unión al ARN/metabolismo , Neoplasias de la Mama/metabolismo , Cadherinas/genética , Línea Celular Tumoral , Proteínas de Unión al ADN , Epilepsia/genética , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Humanos , Discapacidad Intelectual/genética , Mutación , Proteínas Asociadas a Matriz Nuclear/genética , Factores de Transcripción de Octámeros/genética , Linaje , Protocadherinas , Proteínas de Unión al ARN/genéticaRESUMEN
Large numbers of long noncoding RNAs have been discovered in recent years, but only a few have been characterized. NEAT1 (nuclear paraspeckle assembly transcript 1) is a mammalian long noncoding RNA that is important for the reproductive physiology of mice, cancer development, and the formation of subnuclear bodies termed paraspeckles. The two major isoforms of NEAT1 (3.7 kb NEAT1_1 and 23 kb NEAT1_2 in human) are generated from a common promoter and are produced through the use of alternative transcription termination sites. This gene structure has made the functional relationship between the two isoforms difficult to dissect. Here we used CRISPR-Cas9 genome editing to create several different cell lines: total NEAT1 knockout cells, cells that only express the short form NEAT1_1, and cells with twofold more NEAT1_2. Using these reagents, we obtained evidence that NEAT1_1 is not a major component of paraspeckles. In addition, our data suggest NEAT1_1 localizes in numerous nonparaspeckle foci we termed "microspeckles," which may carry paraspeckle-independent functions. This study highlights the complexity of lncRNA and showcases how genome editing tools are useful in dissecting the structural and functional roles of overlapping transcripts.
Asunto(s)
Edición Génica , ARN Largo no Codificante/genética , Sistemas CRISPR-Cas , Línea Celular , Núcleo Celular/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Técnicas de Inactivación de Genes , Humanos , Isoformas de ARN , Factores de Empalme de ARN/metabolismo , Transporte de ARN , ARN Guía de KinetoplastidaRESUMEN
Nuclear proteins are often given a concise title that captures their function, such as 'transcription factor,' 'polymerase' or 'nuclear-receptor.' However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein-protein and protein-nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology.
Asunto(s)
Regulación de la Expresión Génica/genética , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción de Octámeros/metabolismo , Factor de Empalme Asociado a PTB/metabolismo , Procesamiento Postranscripcional del ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Reparación del ADN/genética , Proteínas de Unión al ADN , Drosophila melanogaster , Humanos , Proteínas Asociadas a Matriz Nuclear/ultraestructura , Proteínas Nucleares/ultraestructura , Factores de Transcripción de Octámeros/ultraestructura , Factor de Empalme Asociado a PTB/ultraestructura , Mapas de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas de Unión al ARN/ultraestructura , Transcripción Genética/genética , Activación Transcripcional/genéticaRESUMEN
The field of structural biology has the unique advantage of being able to provide a comprehensive picture of biological mechanisms at the molecular and atomic level. Long noncoding RNAs (lncRNAs) represent the new frontier in the molecular biology of complex organisms yet remain the least characterised of all the classes of RNA. Thousands of new lncRNAs are being reported each year yet very little structural data exists for this rapidly expanding field. The length of lncRNAs ranges from 200 nt to over 100 kb in length and they generally exhibit low cellular abundance. Therefore, obtaining sufficient quantities of lncRNA to use for structural analysis is challenging. However, as technologies develop structures of lncRNAs are starting to emerge providing important information regarding their mechanism of action. Here we review the current methods used to determine the structure of lncRNA and lncRNA:protein complexes and describe the significant contribution structural biology has and will make to the field of lncRNA research. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa.
Asunto(s)
Complejos Multiproteicos/química , Conformación de Ácido Nucleico , ARN Largo no Codificante/química , ARN/química , Humanos , Complejos Multiproteicos/genética , ARN/genética , ARN Largo no Codificante/genéticaRESUMEN
NEAT1 RNA, a highly abundant 4 kb ncRNA, is retained in nuclei in approximately 10 to 20 large foci that we show are completely coincident with paraspeckles, nuclear domains implicated in mRNA nuclear retention. Depletion of NEAT1 RNA via RNAi eradicates paraspeckles, suggesting that it controls sequestration of the paraspeckle proteins PSP1 and p54, factors linked to A-I editing. Unlike overexpression of PSP1, NEAT1 overexpression increases paraspeckle number, and paraspeckles emanate exclusively from the NEAT1 transcription site. The PSP-1 RNA binding domain is required for its colocalization with NEAT1 RNA in paraspeckles, and biochemical analyses support that NEAT1 RNA binds with paraspeckle proteins. Unlike other nuclear-retained RNAs, NEAT1 RNA is not A-I edited, consistent with a structural role in paraspeckles. Collectively, results demonstrate that NEAT1 functions as an essential structural determinant of paraspeckles, providing a precedent for a ncRNA as the foundation of a nuclear domain.
Asunto(s)
Núcleo Celular/metabolismo , Cuerpos de Inclusión Intranucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , ARN Nuclear Pequeño/fisiología , Animales , Células Cultivadas , Proteínas de Cloroplastos , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inmunoprecipitación , Ratones , Interferencia de ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
SFPQ, (a.k.a. PSF), is a human tumor suppressor protein that regulates many important functions in the cell nucleus including coordination of long non-coding RNA molecules into nuclear bodies. Here we describe the first crystal structures of Splicing Factor Proline and Glutamine Rich (SFPQ), revealing structural similarity to the related PSPC1/NONO heterodimer and a strikingly extended structure (over 265 Å long) formed by an unusual anti-parallel coiled-coil that results in an infinite linear polymer of SFPQ dimers within the crystals. Small-angle X-ray scattering and transmission electron microscopy experiments show that polymerization is reversible in solution and can be templated by DNA. We demonstrate that the ability to polymerize is essential for the cellular functions of SFPQ: disruptive mutation of the coiled-coil interaction motif results in SFPQ mislocalization, reduced formation of nuclear bodies, abrogated molecular interactions and deficient transcriptional regulation. The coiled-coil interaction motif thus provides a molecular explanation for the functional aggregation of SFPQ that directs its role in regulating many aspects of cellular nucleic acid metabolism.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Polímeros/química , Proteínas de Unión al ARN/química , Western Blotting , Cristalografía por Rayos X , Ensayo de Cambio de Movilidad Electroforética , Humanos , Microscopía Electrónica de Transmisión , Factor de Empalme Asociado a PTB , Conformación Proteica , Proteínas de Unión al ARN/fisiologíaRESUMEN
Transcriptomic analyses have identified tens of thousands of intergenic, intronic, and cis-antisense long noncoding RNAs (lncRNAs) that are expressed from mammalian genomes. Despite progress in functional characterization, little is known about the post-transcriptional regulation of lncRNAs and their half-lives. Although many are easily detectable by a variety of techniques, it has been assumed that lncRNAs are generally unstable, but this has not been examined genome-wide. Utilizing a custom noncoding RNA array, we determined the half-lives of â¼800 lncRNAs and â¼12,000 mRNAs in the mouse Neuro-2a cell line. We find only a minority of lncRNAs are unstable. LncRNA half-lives vary over a wide range, comparable to, although on average less than, that of mRNAs, suggestive of complex metabolism and widespread functionality. Combining half-lives with comprehensive lncRNA annotations identified hundreds of unstable (half-life < 2 h) intergenic, cis-antisense, and intronic lncRNAs, as well as lncRNAs showing extreme stability (half-life > 16 h). Analysis of lncRNA features revealed that intergenic and cis-antisense RNAs are more stable than those derived from introns, as are spliced lncRNAs compared to unspliced (single exon) transcripts. Subcellular localization of lncRNAs indicated widespread trafficking to different cellular locations, with nuclear-localized lncRNAs more likely to be unstable. Surprisingly, one of the least stable lncRNAs is the well-characterized paraspeckle RNA Neat1, suggesting Neat1 instability contributes to the dynamic nature of this subnuclear domain. We have created an online interactive resource (http://stability.matticklab.com) that allows easy navigation of lncRNA and mRNA stability profiles and provides a comprehensive annotation of ~7200 mouse lncRNAs.
Asunto(s)
Genoma , Ratones/genética , Estabilidad del ARN , ARN no Traducido/metabolismo , Análisis de Varianza , Animales , Línea Celular Tumoral , Análisis por Conglomerados , Expresión Génica , Semivida , Humanos , Anotación de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN no Traducido/genéticaRESUMEN
Proteins of the Drosophila behavior/human splicing (DBHS) family include mammalian SFPQ (PSF), NONO (p54nrb), PSPC1, and invertebrate NONA and Hrp65. DBHS proteins are predominately nuclear, and are involved in transcriptional and posttranscriptional gene regulatory functions as well as DNA repair. DBHS proteins influence a wide gamut of biological processes, including the regulation of circadian rhythm, carcinogenesis, and progression of cancer. Additionally, mammalian DBHS proteins associate with the architectural long noncoding RNA NEAT1 (Menε/ß) to form paraspeckles, subnuclear bodies that alter gene expression via the nuclear retention of RNA. Here we describe the crystal structure of the heterodimer of the multidomain conserved region of the DBHS proteins, PSPC1 and NONO. These proteins form an extensively intertwined dimer, consistent with the observation that the different DBHS proteins are typically copurified from mammalian cells, and suggesting that they act as obligate heterodimers. The PSPC1/NONO heterodimer has a right-handed antiparallel coiled-coil that positions two of four RNA recognition motif domains in an unprecedented arrangement on either side of a 20-Å channel. This configuration is supported by a protein:protein interaction involving the NONA/paraspeckle domain, which is characteristic of the DBHS family. By examining various mutants and truncations in cell culture, we find that DBHS proteins require an additional antiparallel coiled-coil emanating from either end of the dimer for paraspeckle subnuclear body formation. These results suggest that paraspeckles may potentially form through self-association of DBHS dimers into higher-order structures.
Asunto(s)
Espacio Intranuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/química , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Factores de Transcripción de Octámeros/química , Factores de Transcripción de Octámeros/metabolismo , Multimerización de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada/genética , Proteínas de Unión al ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Dominios y Motivos de Interacción de Proteínas , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Paraspeckle nuclear bodies form when the NEAT1 long noncoding RNA is transcribed and bound by multiple RNA-binding proteins. First described 20 years ago, in recent years a growing appreciation of paraspeckle dynamics has led to new understandings, in both structure and function. Structurally, paraspeckles form via distinct physico-chemical domains arising from the composition of key proteins, recruited to different parts of NEAT1. These domains interact, creating a core-shell structured paraspeckle via microphase separation. Functionally, many environmental, chemical, and mechanical triggers can alter paraspeckle abundance, with important consequences depending on the cell type, developmental stage, and trigger identity. Underpinning these insights are new tools for paraspeckle research, including screening assays, proximity-based identification tools, and RNA processing modulators. A picture is emerging of paraspeckles as gene regulatory condensates in many healthy and disease settings. Critically, however, paraspeckle functional importance is generally most apparent when cells and organisms face external stressors.
RESUMEN
At a time when there is a growing public interest in animal welfare, it is critical to have objective means to assess the way that an animal experiences a situation. Objectivity is critical to ensure appropriate animal welfare outcomes. Existing behavioural, physiological, and neurobiological indicators that are used to assess animal welfare can verify the absence of extremely negative outcomes. But welfare is more than an absence of negative outcomes and an appropriate indicator should reflect the full spectrum of experience of an animal, from negative to positive. In this review, we draw from the knowledge of human biomedical science to propose a list of candidate biological markers (biomarkers) that should reflect the experiential state of non-human animals. The proposed biomarkers can be classified on their main function as endocrine, oxidative stress, non-coding molecular, and thermobiological markers. We also discuss practical challenges that must be addressed before any of these biomarkers can become useful to assess the experience of an animal in real-life.