RESUMEN
CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3'-end processing of mRNA precursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5'-3' exonuclease activity in the U7 machinery, degrading the downstream cleavage product after the endonucleolytic cleavage. Recent studies show that CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs, spurring interest in identifying new small-molecule inhibitors against this enzyme. CPSF73 nuclease activity has so far been demonstrated using a gel-based end-point assay, using radiolabeled or fluorescently labeled RNA substrates. By taking advantage of unique properties of the U7 machinery, we have developed a novel, real-time fluorescence assay for the nuclease activity of CPSF73. This assay is facile and high-throughput, and should also be helpful for the discovery of new CPSF73 inhibitors.
Asunto(s)
Bioensayo , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Histonas/metabolismo , Procesamiento de Término de ARN 3' , Precursores del ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/metabolismo , Sistema Libre de Células , Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fluorescencia , Colorantes Fluorescentes/química , Histonas/química , Histonas/genética , Humanos , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacología , Piperazinas/química , Piperazinas/farmacología , Proteolisis , Precursores del ARN/química , Precursores del ARN/genética , Rodaminas/química , Ribonucleoproteína Nuclear Pequeña U7/química , Ribonucleoproteína Nuclear Pequeña U7/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
In animal cells, replication-dependent histone pre-mRNAs are processed at the 3'-end by an endonucleolytic cleavage carried out by the U7 snRNP, a machinery that contains the U7 snRNA and many protein subunits. Studies on the composition of this machinery and understanding of its role in 3'-end processing were greatly facilitated by the development of an in vitro system utilizing nuclear extracts from mammalian cells 35 years ago and later from Drosophila cells. Most recently, recombinant expression and purification of the components of the machinery have enabled the full reconstitution of an active machinery and its complex with a model pre-mRNA substrate, using 13 proteins and 2 RNAs, and the determination of the structure of this active machinery. This chapter presents protocols for preparing nuclear extracts containing endogenous processing machinery, for assembling semi-recombinant and fully reconstituted machineries, and for histone pre-mRNA 3'-end processing assays with these samples.
Asunto(s)
Histonas , Precursores del ARN , Animales , Drosophila/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Ribonucleoproteína Nuclear Pequeña U7/genética , Ribonucleoproteína Nuclear Pequeña U7/metabolismoRESUMEN
Genes encoding replication-dependent histones lack introns, and the mRNAs produced are a unique class of RNA polymerase II transcripts in eukaryotic cells that do not end in a polyadenylated tail. Mature mRNAs are thus formed by a single endonucleolytic cleavage that releases the pre-mRNA from the DNA and is the only processing event necessary. U7 snRNP is one of the key factors that determines the cleavage site within the 3'UTR of replication-dependent histone pre-mRNAs. We have previously showed that the FUS protein interacts with U7 snRNA/snRNP and regulates the expression of histone genes by stimulating transcription and 3' end maturation. Mutations in the FUS gene first identified in patients with amyotrophic lateral sclerosis (ALS) lead to the accumulation of the FUS protein in cytoplasmic inclusions. Here, we report that mutations in FUS lead to disruption of the transcriptional activity of FUS and mislocalization of U7 snRNA/snRNP in cytoplasmic aggregates in cellular models and primary neurons. As a consequence, decreased transcriptional efficiency and aberrant 3' end processing of histone pre-mRNAs were observed. This study highlights for the first time the deregulation of replication-dependent histone gene expression and its involvement in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Regulación de la Expresión Génica , Histonas/metabolismo , Mutación , Proteína FUS de Unión a ARN/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Regiones no Traducidas 3' , Línea Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Perfilación de la Expresión Génica , Células HeLa , Humanos , Hibridación Fluorescente in Situ , Neurociencias , Plásmidos/metabolismo , ARN Nuclear Pequeño/genética , Ribonucleoproteínas Nucleares Pequeñas/genéticaRESUMEN
Inappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA-processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS-stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Interferón Tipo I/biosíntesis , Precursores del ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Enfermedades Autoinmunes del Sistema Nervioso/genética , Enfermedades Autoinmunes del Sistema Nervioso/inmunología , Línea Celular , ADN/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Células HCT116 , Células HEK293 , Enfermedades Autoinflamatorias Hereditarias/genética , Enfermedades Autoinflamatorias Hereditarias/inmunología , Humanos , Proteínas de la Membrana/metabolismo , Malformaciones del Sistema Nervioso/genética , Malformaciones del Sistema Nervioso/inmunología , Nucleótidos Cíclicos/biosíntesis , Nucleotidiltransferasas/metabolismoRESUMEN
Metazoan replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP, an RNA-guided endonuclease that contains U7 snRNA, seven proteins of the Sm ring, FLASH, and four polyadenylation factors: symplekin, CPSF73, CPSF100, and CstF64. A fully recombinant U7 snRNP was recently reconstituted from all 13 components for functional and structural studies and shown to accurately cleave histone pre-mRNAs. Here, we analyzed the activity of recombinant U7 snRNP in more detail. We demonstrate that in addition to cleaving histone pre-mRNAs endonucleolytically, reconstituted U7 snRNP acts as a 5'-3' exonuclease that degrades the downstream product generated from histone pre-mRNAs as a result of the endonucleolytic cleavage. Surprisingly, recombinant U7 snRNP also acts as an endonuclease on single-stranded DNA substrates. All these activities depend on the ability of U7 snRNA to base-pair with the substrate and on the presence of the amino-terminal domain (NTD) of symplekin in either cis or trans, and are abolished by mutations within the catalytic center of CPSF73, or by binding of the NTD to the SSU72 phosphatase of RNA polymerase II. Altogether, our results demonstrate that recombinant U7 snRNP functionally mimics its endogenous counterpart and provide evidence that CPSF73 is both an endonuclease and a 5'-3' exonuclease, consistent with the activity of other members of the ß-CASP family. Our results also raise the intriguing possibility that CPSF73 may be involved in some aspects of DNA metabolism in vivo.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Endonucleasas/genética , Exonucleasas/genética , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Animales , Histonas/genética , Ratones , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN/genéticaRESUMEN
In animal cells, replication-dependent histone pre-mRNAs are cleaved at the 3' end by U7 snRNP consisting of two core components: a â¼60-nucleotide U7 snRNA and a ring of seven proteins, with Lsm10 and Lsm11 replacing the spliceosomal SmD1 and SmD2. Lsm11 interacts with FLASH and together they recruit the endonuclease CPSF73 and other polyadenylation factors, forming catalytically active holo U7 snRNP. Here, we assembled core U7 snRNP bound to FLASH from recombinant components and analyzed its appearance by electron microscopy and ability to support histone pre-mRNA processing in the presence of polyadenylation factors from nuclear extracts. We demonstrate that semi-recombinant holo U7 snRNP reconstituted in this manner has the same composition and functional properties as endogenous U7 snRNP, and accurately cleaves histone pre-mRNAs in a reconstituted in vitro processing reaction. We also demonstrate that the U7-specific Sm ring assembles efficiently in vitro on a spliceosomal Sm site but the engineered U7 snRNP is functionally impaired. This approach offers a unique opportunity to study the importance of various regions in the Sm proteins and U7 snRNA in 3' end processing of histone pre-mRNAs.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Secuencia de Aminoácidos/genética , Animales , Núcleo Celular/genética , Drosophila/genética , Histonas/genética , Humanos , Ratones , Unión Proteica/genética , Precursores del ARN/genética , Empalmosomas/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The RNA-binding protein ALYREF plays key roles in nuclear export and also 3'-end processing of polyadenylated mRNAs, but whether such regulation also extends to non-polyadenylated RNAs is unknown. Replication-dependent (RD)-histone mRNAs are not polyadenylated, but instead end in a stem-loop (SL) structure. Here, we demonstrate that ALYREF prevalently binds a region next to the SL on RD-histone mRNAs. SL-binding protein (SLBP) directly interacts with ALYREF and promotes its recruitment. ALYREF promotes histone pre-mRNA 3'-end processing by facilitating U7-snRNP recruitment through physical interaction with the U7-snRNP-specific component Lsm11. Furthermore, ALYREF, together with other components of the TREX complex, enhances histone mRNA export. Moreover, we show that 3'-end processing promotes ALYREF recruitment and histone mRNA export. Together, our results point to an important role of ALYREF in coordinating 3'-end processing and nuclear export of non-polyadenylated mRNAs.
Asunto(s)
Histonas/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento Postranscripcional del ARN , Transporte de ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Histonas/genética , Humanos , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Factores de Transcripción/genéticaRESUMEN
The scaffolding protein Symplekin is part of multiple complexes involved in generating and modifying the 3' end of mRNAs, including cleavage-polyadenylation, histone pre-mRNA processing and cytoplasmic polyadenylation. To study these functions in vivo, we examined the localization of Symplekin during development and generated mutations of the Drosophila Symplekin gene. Mutations in Symplekin that reduce Symplekin protein levels alter the efficiency of both poly A(+) and histone mRNA 3' end formation resulting in lethality or sterility. Histone mRNA synthesis takes place at the histone locus body (HLB) and requires a complex composed of Symplekin and several polyadenylation factors that associates with the U7 snRNP. Symplekin is present in the HLB in the early embryo when Cyclin E/Cdk2 is active and histone genes are expressed and is absent from the HLB in cells that have exited the cell cycle. During oogenesis, Symplekin is preferentially localized to HLBs during S-phase in endoreduplicating follicle cells when histone mRNA is synthesized. After the completion of endoreplication, Symplekin accumulates in the cytoplasm, in addition to the nucleoplasm, and localizes to tricellular junctions of the follicle cell epithelium. This localization depends on the RNA binding protein ypsilon schachtel. CPSF-73 and a number of mRNAs are localized at this same site, suggesting that Symplekin participates in cytoplasmic polyadenylation at tricellular junctions.
Asunto(s)
Proteínas de Drosophila/biosíntesis , Desarrollo Embrionario , Histonas/genética , Poliadenilación/genética , Factores de Escisión y Poliadenilación de ARNm/biosíntesis , Animales , Citoplasma/genética , Drosophila , Proteínas de Drosophila/genética , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Mutación , Nucleoplasminas/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Fase S/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The 3' end processing of animal replication-dependent histone mRNAs is activated during G1/S-phase transition. The processing site is recognized by stem-loop binding protein and the U7 snRNP, but cleavage additionally requires a heat-labile factor (HLF), composed of cleavage/polyadenylation specificity factor, symplekin, and cleavage stimulation factor 64 (CstF64). Although HLF has been shown to be cell cycle regulated, the mechanism of this regulation is unknown. Here we show that levels of CstF64 increase toward the S phase and its depletion affects histone RNA processing, S-phase progression, and cell proliferation. Moreover, analyses of the interactions between CstF64, symplekin, and the U7 snRNP-associated proteins FLASH and Lsm11 indicate that CstF64 is important for recruiting HLF to histone precursor mRNA (pre-mRNA)-resident proteins. Thus, CstF64 is central to the function of HLF and appears to be at least partly responsible for its cell cycle regulation. Additionally, we show that misprocessed histone transcripts generated upon CstF64 depletion mainly accumulate in the nucleus, where they are targets of the exosome machinery, while a small cytoplasmic fraction is partly associated with polysomes.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Histonas/genética , Proteínas Nucleares/genética , Procesamiento de Término de ARN 3'/genética , Proteínas de Unión al ARN/fisiología , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Proliferación Celular , Factor de Estimulación del Desdoblamiento , Células HEK293 , Células HeLa , Humanos , Precursores del ARN/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Fase S/genéticaRESUMEN
3'-End cleavage of animal replication-dependent histone pre-mRNAs is controlled by the U7 snRNP. Lsm11, the largest component of the U7-specific Sm ring, interacts with FLASH, and in mammalian nuclear extracts these two proteins form a platform that recruits the CPSF73 endonuclease and other polyadenylation factors to the U7 snRNP. FLASH is limiting, and the majority of the U7 snRNP in mammalian extracts exists as a core particle consisting of the U7 snRNA and the Sm ring. Here, we purified the U7 snRNP from Drosophila nuclear extracts and characterized its composition by mass spectrometry. In contrast to the mammalian U7 snRNP, a significant fraction of the Drosophila U7 snRNP contains endogenous FLASH and at least six subunits of the polyadenylation machinery: symplekin, CPSF73, CPSF100, CPSF160, WDR33, and CstF64. The same composite U7 snRNP is recruited to histone pre-mRNA for 3'-end processing. We identified a motif in Drosophila FLASH that is essential for the recruitment of the polyadenylation complex to the U7 snRNP and analyzed the role of other factors, including SLBP and Ars2, in 3'-end processing of Drosophila histone pre-mRNAs. SLBP that binds the upstream stem-loop structure likely recruits a yet-unidentified essential component(s) to the processing machinery. In contrast, Ars2, a protein previously shown to interact with FLASH in mammalian cells, is dispensable for processing in Drosophila. Our studies also demonstrate that Drosophila symplekin and three factors involved in cleavage and polyadenylation-CPSF, CstF, and CF Im-are present in Drosophila nuclear extracts in a stable supercomplex.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/genética , Procesamiento de Término de ARN 3' , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Drosophila melanogaster , Histonas/metabolismo , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Mapeo de Interacción de Proteínas , Subunidades de Proteína/metabolismo , División del ARN , Precursores del ARN/genética , ARN Mensajero/genética , Ribonucleoproteína Nuclear Pequeña U7/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Animal replication-dependent histone pre-mRNAs are processed at the 3' end by endonucleolytic cleavage that is not followed by polyadenylation. The cleavage reaction is catalyzed by CPSF73 and depends on the U7 snRNP and its integral component, Lsm11. A critical role is also played by the 220-kDa protein FLASH, which interacts with Lsm11. Here we demonstrate that the N-terminal regions of these two proteins form a platform that tightly interacts with a unique combination of polyadenylation factors: symplekin, CstF64, and all CPSF subunits, including the endonuclease CPSF73. The interaction is inhibited by alterations in each component of the FLASH/Lsm11 complex, including point mutations in FLASH that are detrimental for processing. The same polyadenylation factors are associated with the endogenous U7 snRNP and are recruited in a U7-dependent manner to histone pre-mRNA. Collectively, our studies identify the molecular mechanism that recruits the CPSF73 endonuclease to histone pre-mRNAs, reveal an unexpected complexity of the U7 snRNP, and suggest that in animal cells polyadenylation factors assemble into two alternative complexes-one specifically crafted to generate polyadenylated mRNAs and the other to generate nonpolyadenylated histone mRNAs that end with the stem-loop.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Histonas/metabolismo , Procesamiento de Término de ARN 3' , Precursores del ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencias de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Secuencia de Bases , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Estimulación del Desdoblamiento , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Metazoan replication-dependent histone mRNAs are the only nonpolyadenylated cellular mRNAs. Formation of the histone mRNA 3' end requires the U7 snRNP, which contains Lsm10 and Lsm11, and FLASH, a processing factor that binds Lsm11. Here, we identify sequences in Drosophila FLASH (dFLASH) that bind Drosophila Lsm11 (dLsm11), allow localization of dFLASH to the nucleus and histone locus body (HLB), and participate in histone pre-mRNA processing in vivo. Amino acids 105-154 of dFLASH bind to amino acids 1-78 of dLsm11. A two-amino acid mutation of dLsm11 that prevents dFLASH binding but does not affect localization of U7 snRNP to the HLB cannot rescue the lethality or histone pre-mRNA processing defects resulting from an Lsm11 null mutation. The last 45 amino acids of FLASH are required for efficient localization to the HLB in Drosophila cultured cells. Removing the first 64 amino acids of FLASH has no effect on processing in vivo. Removal of 13 additional amino acids of dFLASH results in a dominant negative protein that binds Lsm11 but inhibits processing of histone pre-mRNA in vivo. Inhibition requires the Lsm11 binding site, suggesting that the mutant dFLASH protein sequesters the U7 snRNP in an inactive complex and that residues between 64 and 77 of dFLASH interact with a factor required for processing. Together, these studies demonstrate that direct interaction between dFLASH and dLsm11 is essential for histone pre-mRNA processing in vivo and for proper development and viability in flies.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/genética , Histonas/genética , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/genética , Células Cultivadas , Drosophila/metabolismo , Proteínas de Drosophila/genética , Histonas/metabolismo , Procesamiento Postranscripcional del ARN , ARN Nuclear Heterogéneo/genética , ARN Nuclear Heterogéneo/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/genética , Ribonucleoproteína Nuclear Pequeña U7/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genéticaRESUMEN
BACKGROUND: Octamer-binding factor 6 (Oct-6, Pou3f1, SCIP, Tst-1) is a transcription factor of the Pit-Oct-Unc (POU) family. POU proteins regulate key developmental processes and have been identified from a diverse range of species. Oct-6 expression is described to be confined to the developing brain, Schwann cells, oligodendrocyte precursors, testes, and skin. Its function is primarily characterised in Schwann cells, where it is required for correctly timed transition to the myelinating state. In the present study, we report that Oct-6 is an interferon (IFN)-inducible protein and show for the first time expression in murine fibroblasts and macrophages. RESULTS: Oct-6 was induced by type I and type II IFN, but not by interleukin-6. Induction of Oct-6 after IFNbeta treatment was mainly dependent on signal transducer and activator of transcription 1 (Stat1) and partially on tyrosine kinase 2 (Tyk2). Chromatin immunopreciptitation experiments revealed binding of Stat1 to the Oct-6 promoter in a region around 500 bp upstream of the transcription start site, a region different from the downstream regulatory element involved in Schwann cell-specific Oct-6 expression. Oct-6 was also induced by dsRNA treatment and during viral infections, in both cases via autocrine/paracrine actions of IFNalpha/beta. Using microarray and RT-qPCR, we furthermore show that Oct-6 is involved in the regulation of transcriptional responses to dsRNA, in particular in the gene regulation of serine/threonine protein kinase 40 (Stk40) and U7 snRNA-associated Sm-like protein Lsm10 (Lsm10). CONCLUSION: Our data show that Oct-6 expression is not as restricted as previously assumed. Induction of Oct-6 by IFNs and viruses in at least two different cell types, and involvement of Oct-6 in gene regulation after dsRNA treatment, suggest novel functions of Oct-6 in innate immune responses.
Asunto(s)
Fibroblastos/metabolismo , Macrófagos/metabolismo , Factor 6 de Transcripción de Unión a Octámeros/metabolismo , Virosis/metabolismo , Animales , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibroblastos/virología , Inmunidad Innata/genética , Interferón beta/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/patología , Macrófagos/virología , Ratones , Ratones Noqueados , Análisis por Micromatrices , Morfogénesis/genética , Factor 6 de Transcripción de Unión a Octámeros/genética , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/genética , ARN Bicatenario/farmacología , ARN Viral/farmacología , Ribonucleoproteína Nuclear Pequeña U7/biosíntesis , Ribonucleoproteína Nuclear Pequeña U7/genética , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Activación Transcripcional/efectos de los fármacos , Virosis/genética , Virosis/inmunologíaRESUMEN
Metazoan replication-dependent histone mRNAs are not polyadenylated, and instead terminate in a conserved stem-loop structure generated by an endonucleolytic cleavage of the pre-mRNA involving U7 snRNP. U7 snRNP contains two like-Sm proteins, Lsm10 and Lsm11, which replace SmD1 and SmD2 in the canonical heptameric Sm protein ring that binds spliceosomal snRNAs. Here we show that mutations in either the Drosophila Lsm10 or the Lsm11 gene disrupt normal histone pre-mRNA processing, resulting in production of poly(A)+ histone mRNA as a result of transcriptional read-through to cryptic polyadenylation sites present downstream from each histone gene. This molecular phenotype is indistinguishable from that which we previously described for mutations in U7 snRNA. Lsm10 protein fails to accumulate in Lsm11 mutants, suggesting that a pool of Lsm10-Lsm11 dimers provides precursors for U7 snRNP assembly. Unexpectedly, U7 snRNA was detected in Lsm11 and Lsm1 mutants and could be precipitated with anti-trimethylguanosine antibodies, suggesting that it assembles into a snRNP particle in the absence of Lsm10 and Lsm11. However, this U7 snRNA could not be detected at the histone locus body, suggesting that Lsm10 and Lsm11 are necessary for U7 snRNP localization. In contrast to U7 snRNA null mutants, which are viable, Lsm10 and Lsm11 mutants do not survive to adulthood. Because we cannot detect differences in the histone mRNA phenotype between Lsm10 or Lsm11 and U7 mutants, we propose that the different terminal developmental phenotypes result from the participation of Lsm10 and Lsm11 in an essential function that is distinct from histone pre-mRNA processing and that is independent of U7 snRNA.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/crecimiento & desarrollo , Drosophila/genética , Histonas/genética , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/fisiología , Ribonucleoproteínas Nucleares Pequeñas/fisiología , Animales , Animales Modificados Genéticamente , Drosophila/metabolismo , Proteínas de Drosophila/genética , Femenino , Fertilidad/genética , Genes del Desarrollo/fisiología , Genes Letales/genética , Histonas/metabolismo , Masculino , Mutación/fisiología , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , Ribonucleoproteína Nuclear Pequeña U7/genética , Ribonucleoproteínas Nucleares Pequeñas/genéticaRESUMEN
Cleavage of histone pre-mRNAs at the 3' end is guided by the U7 snRNP, which is a component of a larger 3'-end processing complex. To identify other components of this complex, we isolated proteins that stably associate with a fragment of histone pre-mRNA containing all necessary processing elements and a biotin affinity tag at the 5' end. Among the isolated proteins, we identified three well-characterized processing factors: the stem-loop binding protein (SLBP), which interacts with the stem-loop structure upstream of the cleavage site, and both Lsm11 and SmB, which are components of the U7-specific Sm ring. We also identified 3'hExo/Eri-1, a multifunctional 3' exonuclease that is known to trim the 3' end of 5.8S rRNA. 3'hExo primarily binds to the downstream portion of the stem-loop structure in mature histone mRNA, with the upstream portion being occupied by SLBP. The two proteins bind their respective RNA sites in a cooperative manner, and 3'hExo can recruit SLBP to a mutant stem-loop that itself does not interact with SLBP. UV-cross-linking studies used to characterize interactions within the processing complex demonstrated that 3'hExo also interacts in a U7-dependent manner with unprocessed histone pre-mRNA. However, this interaction is not required for the cleavage reaction. The region between the cleavage site and the U7-binding site interacts with three low-molecular-weight proteins, which were identified as components of the U7-specific Sm core: SmB, SmD3, and Lsm10. These proteins likely rigidify the substrate and function as the molecular ruler in determining the site of cleavage.
Asunto(s)
Histonas/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , Ribonucleoproteína Nuclear Pequeña U7/metabolismo , Regiones no Traducidas 3'/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Línea Celular Tumoral , Inmunoprecipitación , Ratones , Conformación de Ácido Nucleico , Unión Proteica , Precursores del ARN/genética , ARN Lider Empalmado/genética , ARN Lider Empalmado/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/química , Ribonucleoproteína Nuclear Pequeña U7/genéticaRESUMEN
The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.
Asunto(s)
Regiones no Traducidas 5' , Evolución Molecular , Histonas/genética , ARN Mensajero/genética , Animales , Eucariontes/genética , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Protozoario/genética , Ribonucleoproteína Nuclear Pequeña U7/genéticaRESUMEN
In amphibian oocytes, most lateral loops of the lampbrush chromosomes correspond to active transcriptional sites for RNA polymerase II. We show that newly assembled small nuclear ribonucleoprotein (RNP [snRNP]) particles, which are formed upon cytoplasmic injection of fluorescently labeled spliceosomal small nuclear RNAs (snRNAs), target the nascent transcripts of the chromosomal loops. With this new targeting assay, we demonstrate that nonfunctional forms of U1 and U2 snRNAs still associate with the active transcriptional units. In particular, we find that their association with nascent RNP fibrils is independent of their base pairing with pre-messenger RNAs. Additionally, stem loop I of the U1 snRNA is identified as a discrete domain that is both necessary and sufficient for association with nascent transcripts. Finally, in oocytes deficient in splicing, the recruitment of U1, U4, and U5 snRNPs to transcriptional units is not affected. Collectively, these data indicate that the recruitment of snRNPs to nascent transcripts and the assembly of the spliceosome are uncoupled events.
Asunto(s)
ARN Polimerasa II/metabolismo , Precursores del ARN/metabolismo , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/fisiología , Animales , Femenino , Conformación de Ácido Nucleico , ARN Polimerasa II/genética , Precursores del ARN/genética , Empalme del ARN , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/genética , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Ribonucleoproteína Nuclear Pequeña U7/genética , Ribonucleoproteína Nuclear Pequeña U7/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Empalmosomas/genética , XenopusRESUMEN
The organization and molecular composition of complicated Cajal bodies (CBs) and interchromatin granule clusters (IGCs) in oocytes of the house cricket, Acheta domesticus, were studied using immunofluorescent/confocal and Immunogold labeling/electron microscopy. In A. domesticus oocytes, the CB consists of the fibrillar matrix and a central cavity containing a predominantly granular body with insertions of tightly packed fibrillar material. The latter structure was identified as an "internal" IGC, since it is enriched with the SC35 protein, a marker for IGCs. The IGCs located outside the CB were also identified. Microinjections of the fluorescein-tagged U7 snRNA into the ooplasm showed the targeting of the U7 to the matrix of the CB. Some other essential CB components (coilin, snRNPs, fibrillarin) were found to be colocalized in the matrix of the CB. Neither confocal nor Immunogold microscopy revealed significant amounts of RNA polymerase II (pol II) in the CB of A. domesticus oocytes. The splicing factor SC35 was detected in the matrix of the CB. In oocytes treated with DRB, the amount of IGCs in the nucleoplasm increased significantly, granular and fibrillar components of IGCs were segregated, and the fibrillar areas accumulated pol II. Additionally, IG-like granules were shown to display on the surface of the CB probably due to a shifting from the internal IGC. We believe that in A. domesticus oocytes, CBs are involved in nuclear distribution of splicing factors, but their role in pol II transport is less significant. We also suggest that the formation of complicated CBs is a result of interconnection between two different nuclear domains, CBs and IGCs.
Asunto(s)
Núcleo Celular/ultraestructura , Cromatina/metabolismo , Cuerpos Enrollados/metabolismo , Cuerpos Enrollados/ultraestructura , Gryllidae/ultraestructura , Oocitos/ultraestructura , Animales , Núcleo Celular/metabolismo , Cromatina/ultraestructura , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Gryllidae/metabolismo , Microscopía Confocal , Proteínas Nucleares/metabolismo , Oocitos/metabolismo , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U7/química , Ribonucleoproteína Nuclear Pequeña U7/genética , Ribonucleoproteínas/metabolismo , Factores de Empalme Serina-Arginina , Fracciones Subcelulares/ultraestructura , Distribución TisularRESUMEN
Formation of the 3' end of replication-dependent histone mRNAs is most robust during S phase and is mediated by both the stem-loop binding protein (SLBP) and the U7 snRNP. We previously identified a 100-kDa zinc finger protein (ZFP100) as a component of U7 snRNP that interacts with the SLBP/pre-mRNA complex. Here, we show that myc- or GFP-tagged ZFP100 overexpressed after transfection is concentrated in Cajal bodies (CBs), and unlike components of the spliceosomal snRNPs, photobleaching experiments demonstrate that ZFP100 is stably associated with CBs. Of the 18 zinc fingers contained within ZFP100, the region encompassing fingers 2-6 is sufficient to maintain CB localization. Zn fingers 5-10 are required for maximal binding of ZFP100 to a 20-amino-acid region of Lsm11, a U7 snRNP core protein. Expression of ZFP100 stimulates histone mRNA processing in vivo, assayed by activation of a reporter gene that encodes a GFP mRNA ending in a histone 3' end. Importantly, the domain that is required for CB localization and Lsm11 binding is also sufficient to stimulate histone pre-mRNA processing in vivo. Comparisons with other mammalian ZFP100 orthologs show that the central Zn fingers sufficient for in vivo activity are most highly conserved, whereas the number and sequence of the Zn fingers in the N- and C-terminal domains vary.
Asunto(s)
Proteínas de Unión al ADN/genética , Ribonucleoproteína Nuclear Pequeña U7/química , Dedos de Zinc , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Cuerpos Enrollados/ultraestructura , Secuencia Conservada , Proteínas de Unión al ADN/química , Citometría de Flujo , Eliminación de Gen , Células HeLa , Histonas/genética , Humanos , Datos de Secuencia Molecular , Plásmidos , Ribonucleoproteína Nuclear Pequeña U7/genéticaRESUMEN
Cajal bodies (CBs) are nuclear organelles that are usually identified by the marker protein p80-coilin. Because no orthologue of coilin is known in Drosophila melanogaster, we identified D. melanogaster CBs using probes for other components that are relatively diagnostic for CBs in vertebrate cells. U85 small CB-specific RNA, U2 small nuclear RNA, the survival of motor neurons protein, and fibrillarin occur together in a nuclear body that is closely associated with the nucleolus. Based on its similarity to CBs in other organisms, we refer to this structure as the D. melanogaster CB. Surprisingly, the D. melanogaster U7 small nuclear RNP resides in a separate nuclear body, which we call the histone locus body (HLB). The HLB is invariably colocalized with the histone gene locus. Thus, canonical CB components are distributed into at least two nuclear bodies in D. melanogaster. The identification of these nuclear bodies now permits a broad range of questions to be asked about CB structure and function in a genetically tractable organism.
