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1.
RNA ; 27(9): 1102-1125, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34187903

RESUMO

Polyadenylated nuclear (PAN) RNA is a long noncoding transcript involved in Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have previously shown that PAN RNA has dynamic secondary structure and protein binding profiles that can be influenced by epitranscriptomic modifications. N 6-methyladenosine (m6A) is one of the most abundant chemical signatures found in viral RNA genomes and virus-encoded RNAs. Here, we combined antibody-independent next-generation mapping with direct RNA sequencing to address the epitranscriptomic status of PAN RNA in KSHV infected cells. We showed that PAN m6A status is dynamic, reaching the highest number of modifications at the late lytic stages of KSHV infection. Using a newly developed method, termed selenium-modified deoxythymidine triphosphate (SedTTP)-reverse transcription (RT) and ligation assisted PCR analysis of m6A (SLAP), we gained insight into the fraction of modification at identified sites. By applying comprehensive proteomic approaches, we identified writers and erasers that regulate the m6A status of PAN, and readers that can convey PAN m6A phenotypic effects. We verified the temporal and spatial subcellular availability of the methylome components for PAN modification by performing confocal microscopy analysis. Additionally, the RNA biochemical probing (SHAPE-MaP) outlined local and global structural alterations invoked by m6A in the context of full-length PAN RNA. This work represents the first comprehensive overview of the dynamic interplay that takes place between the cellular epitranscriptomic machinery and a specific viral RNA in the context of KSHV infected cells.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética , Herpesvirus Humano 8/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Nuclear/genética , Adenosina/genética , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Endonucleases/genética , Endonucleases/metabolismo , Herpesvirus Humano 8/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Linfócitos/metabolismo , Linfócitos/virologia , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Reversa , Análise de Sequência de RNA , Transcriptoma
2.
RNA ; 27(9): 991-1003, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108230

RESUMO

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.


Assuntos
Processamento Alternativo , Proteínas Argonauta/genética , Fatores de Iniciação em Eucariotos/genética , MicroRNAs/genética , RNA Nuclear/genética , Proteínas Argonauta/deficiência , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Fatores de Iniciação em Eucariotos/deficiência , Éxons , Células HCT116 , Humanos , Imunoprecipitação , Íntrons , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Oligorribonucleotídeos/genética , Oligorribonucleotídeos/metabolismo , Ligação Proteica , RNA Nuclear/metabolismo , Análise de Sequência de RNA
3.
EMBO J ; 40(12): e106357, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33938020

RESUMO

The functions of long RNAs, including mRNAs and long noncoding RNAs (lncRNAs), critically depend on their subcellular localization. The identity of the sequences that dictate subcellular localization and their high-resolution anatomy remain largely unknown. We used a suite of massively parallel RNA assays and libraries containing thousands of sequence variants to pinpoint the functional features within the SIRLOIN element, which dictates nuclear enrichment through hnRNPK recruitment. In addition, we profiled the endogenous SIRLOIN RNA-nucleoprotein complex and identified the nuclear RNA-binding proteins SLTM and SNRNP70 as novel SIRLOIN binders. Taken together, using massively parallel assays, we identified the features that dictate binding of hnRNPK, SLTM, and SNRNP70 to SIRLOIN and found that these factors are jointly required for SIRLOIN activity. Our study thus provides a roadmap for high-throughput dissection of functional sequence elements in long RNAs.


Assuntos
RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Humanos , Células MCF-7 , Ligação Proteica , RNA-Seq
4.
J Virol ; 95(13): e0009621, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33853955

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic human gammaherpesvirus and the causative agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). During reactivation, viral genes are expressed in a temporal manner. These lytic genes encode transactivators, core replication proteins, or structural proteins. During reactivation, other viral factors that are required for lytic replication are expressed. The most abundant viral transcript is the long noncoding RNA (lncRNA) known as polyadenylated nuclear (PAN) RNA. lncRNAs have diverse functions, including the regulation of gene expression and the immune response. PAN possesses two main cis-acting elements, the Mta response element (MRE) and the expression and nuclear retention element (ENE). While PAN has been demonstrated to be required for efficient viral replication, the function of these elements within PAN remains unclear. Our goal was to determine if the ENE of PAN is required in the context of infection. A KSHV bacmid containing a deletion of the 79-nucleotide (nt) ENE in PAN was generated to assess the effects of the ENE during viral replication. Our studies demonstrated that the ENE is not required for viral DNA synthesis, lytic gene expression, or the production of infectious virus. Although the ENE is not required for viral replication, we found that the ENE functions to retain PAN in the nucleus, and the absence of the ENE results in an increased accumulation of PAN in the cytoplasm. Furthermore, open reading frame 59 (ORF59), LANA, ORF57, H1.4, and H2A still retain the ability to bind to PAN in the absence of the ENE. Together, our data highlight how the ENE affects the nuclear retention of PAN but ultimately does not play an essential role during lytic replication. Our data suggest that PAN may have other functional domains apart from the ENE. IMPORTANCE KSHV is an oncogenic herpesvirus that establishes latency and exhibits episodes of reactivation. KSHV disease pathologies are most often associated with the lytic replication of the virus. PAN RNA is the most abundant viral transcript during the reactivation of KSHV and is required for viral replication. Deletion and knockdown of PAN resulted in defects in viral replication and reduced virion production in the absence of PAN RNA. To better understand how the cis elements within PAN may contribute to its function, we investigated if the ENE of PAN was necessary for viral replication. Although the ENE had previously been extensively studied with both biochemical and in vitro approaches, this is the first study to demonstrate the role of the ENE in the context of infection and that the ENE of PAN is not required for the lytic replication of KSHV.


Assuntos
Regulação Viral da Expressão Gênica/genética , Herpesvirus Humano 8/crescimento & desenvolvimento , Herpesvirus Humano 8/genética , RNA Longo não Codificante/genética , Ativação Viral/genética , Latência Viral/genética , Hiperplasia do Linfonodo Gigante/virologia , Linhagem Celular Tumoral , Células HEK293 , Herpesvirus Humano 8/fisiologia , Humanos , RNA Mensageiro/genética , RNA Nuclear/genética , Sarcoma de Kaposi/virologia , Replicação Viral/genética
5.
Parasitology ; 148(10): 1219-1222, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33729118

RESUMO

Transfer RNAs play a key role in protein synthesis. Following transcription, tRNAs are extensively processed prior to their departure from the nucleus to become fully functional during translation. This includes removal of 5' leaders and 3' trailers by a specific endo- and/or exonuclease, 3' CCA tail addition, posttranscriptional modifications and in some cases intron removal. In this minireview, the critical factors of nuclear tRNA trafficking are described based on studies in classical models such as yeast and human cell lines. In addition, recent findings and identification of novel regulatory loops of nuclear tRNA trafficking in trypanosomes are discussed with emphasis on tRNA modifications. The comparison between the representatives of opisthokonts and excavates serves here to understand the evolutionary conservation and diversity of nuclear tRNA export mechanisms.


Assuntos
RNA Nuclear/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Trypanosoma/metabolismo , Linhagem Celular , Humanos , RNA Nuclear/genética , RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Trypanosoma/genética
6.
Chromosoma ; 130(1): 75-90, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33585981

RESUMO

Within the pericentric regions of human chromosomes reside large arrays of tandemly repeated satellite sequences. Expression of the human pericentric satellite HSATII is prevented by extensive heterochromatin silencing in normal cells, yet in many cancer cells, HSATII RNA is aberrantly expressed and accumulates in large nuclear foci in cis. Expression and aggregation of HSATII RNA in cancer cells is concomitant with recruitment of key chromatin regulatory proteins including methyl-CpG binding protein 2 (MeCP2). While HSATII expression has been observed in a wide variety of cancer cell lines and tissues, the effect of its expression is unknown. We tested the effect of stable expression of HSATII RNA within cells that do not normally express HSATII. Ectopic HSATII expression in HeLa and primary fibroblast cells leads to focal accumulation of HSATII RNA in cis and triggers the accumulation of MeCP2 onto nuclear HSATII RNA bodies. Further, long-term expression of HSATII RNA leads to cell division defects including lagging chromosomes, chromatin bridges, and other chromatin defects. Thus, expression of HSATII RNA in normal cells phenocopies its nuclear accumulation in cancer cells and allows for the characterization of the cellular events triggered by aberrant expression of pericentric satellite RNA.


Assuntos
Divisão Celular , Cromatina/genética , DNA Satélite/genética , Expressão Ectópica do Gene , Proteína 2 de Ligação a Metil-CpG/metabolismo , RNA Nuclear/genética , Células HeLa , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , RNA Longo não Codificante
7.
Parasitology ; 148(10): 1196-1218, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33461637

RESUMO

The passage of mRNAs through the nuclear pores into the cytoplasm is essential in all eukaryotes. For regulation, mRNA export is tightly connected to the full machinery of nuclear mRNA processing, starting at transcription. Export competence of pre-mRNAs gradually increases by both transient and permanent interactions with multiple RNA processing and export factors. mRNA export is best understood in opisthokonts, with limited knowledge in plants and protozoa. Here, I review and compare nuclear mRNA processing and export between opisthokonts and Trypanosoma brucei. The parasite has many unusual features in nuclear mRNA processing, such as polycistronic transcription and trans-splicing. It lacks several nuclear complexes and nuclear-pore-associated proteins that in opisthokonts play major roles in mRNA export. As a consequence, trypanosome mRNA export control is not tight and export can even start co-transcriptionally. Whether trypanosomes regulate mRNA export at all, or whether leakage of immature mRNA to the cytoplasm is kept to a low level by a fast kinetics of mRNA processing remains to be investigated. mRNA export had to be present in the last common ancestor of eukaryotes. Trypanosomes are evolutionary very distant from opisthokonts and a comparison helps understanding the evolution of mRNA export.


Assuntos
RNA Mensageiro/metabolismo , RNA Nuclear/metabolismo , Trypanosoma/metabolismo , Eucariotos
8.
Cancer Commun (Lond) ; 41(2): 140-153, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33455092

RESUMO

BACKGROUND: Small RNAs (sRNAs) extensively mediate gene-specific chromatin regulation in lower organisms. As a dominant type of functional sRNAs in mature mammals, microRNAs mainly regulate gene expression at post-transcription level in the cytoplasm. Currently, whether there exists a type of nuclear-localized sRNAs mediating gene-specific epigenetic regulation in mature mammalian cells remains largely unclear. Here, we profiled sRNAs enriched in the nucleus and investigated their function in mediating gene-specific epigenetic regulation in anti-tumor immunity. METHODS: We established cytoplasmic and nuclear transcriptomes of sRNAs of dendritic cells (DCs) using high-throughput sequencing. Transcription abundances of sRNAs and mRNAs were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay. The associations between sRNAs and Argonaute (AGO) proteins were detected by RNA immunoprecipitation analysis. Synthesized sRNAs and locked nucleic acid (LNA) -modified sRNA inhibitors were used to screen the function of sRNAs in innate immune cells. The effect of sRNA on the enrichment of either chromatin remodeler or histone modification at the gene promoter was analyzed by chromatin immunoprecipitation (ChIP)-qPCR assay. Chromatin accessibility qPCR assay was used to detect the accessibility of gene promoters. A B16 melanoma-bearing mouse model was established to determine the function of sRNAs in tumor-associated macrophages (TAMs) and their effect on tumor growth. RESULTS: We identified a new class of nucleus-localized sRNAs, named snRNA/snoRNA-derived nuclear RNAs (sdnRNAs). Some sdnRNAs were Dicer-independent and had no association with Argonaute proteins. sdnRNA-3, the most abundant Dicer-independent sdnRNAs identified in our analysis, was selected as a representative to examine the biological function of sdnRNAs. sdnRNA-3 selectively inhibited the transcription of Nos2 in macrophages during innate immune response by repressing the chromatin accessibility at Nos2 gene promoter. sdnRNA-3 promoted the enrichments of repressive chromatin-remodeling regulator Mi-2ß and the repressive histone modification H3K27me3 at Nos2 gene promoter. In the B16 melanoma mouse model, we found higher expression of sdnRNA-3 in M2 TAMs than M1 TAMs and DCs. Transfer of sdnRNA-3-silenced macrophages inhibited tumor growth with increased expression of inducible nitric oxide synthase (iNOS) in TAMs. CONCLUSIONS: Our results demonstrated that the sdnRNA-3 repressed the transcription of Nos2 by repressing chromatin accessibility at the promoter, providing new insights into the regulation of macrophage function in tumor immunity.


Assuntos
RNA Nuclear , RNA Nucleolar Pequeno , Macrófagos Associados a Tumor , Animais , Epigênese Genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo
9.
Essays Biochem ; 64(6): 895-905, 2020 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-32885820

RESUMO

In eukaryotes, RNAs newly synthesized by RNA polymerase II (RNAPII) undergo several processing steps prior to transport to the cytoplasm. It has long been known that RNAs with defects in processing or export are removed in the nucleus. Recent studies revealed that RNAs without apparent defects are also subjected to nuclear degradation, indicating that nuclear RNA fate is determined in a more complex and dynamic way than previously thought. Nuclear RNA sorting directly determines the quality and quantity of RNA pools for future translation and thus is of significant importance. In this essay, we will summarize recent studies on this topic, mainly focusing on findings in mammalian system, and discuss about important remaining questions and possible biological relevance for nuclear RNA fate determination.


Assuntos
Núcleo Celular/metabolismo , RNA Nuclear/genética , RNA Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Citoplasma/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Biossíntese de Proteínas , Estabilidade de RNA , Transporte de RNA
10.
Elife ; 92020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32804637

RESUMO

Nuclear RNAi provides a highly tractable system to study RNA-mediated chromatin changes and epigenetic inheritance. Recent studies have indicated that the regulation and function of nuclear RNAi-mediated heterochromatin are highly complex. Our knowledge of histone modifications and the corresponding histonemodifying enzymes involved in the system remains limited. In this study, we show that the heterochromatin mark, H3K23me3, is induced by nuclear RNAi at both exogenous and endogenous targets in C. elegans. In addition, dsRNA-induced H3K23me3 can persist for multiple generations after the dsRNA exposure has stopped. We demonstrate that the histone methyltransferase SET-32, methylates H3K23 in vitro. Both set-32 and the germline nuclear RNAi Argonaute, hrde-1, are required for nuclear RNAi-induced H3K23me3 in vivo. Our data poise H3K23me3 as an additional chromatin modification in the nuclear RNAi pathway and provides the field with a new target for uncovering the role of heterochromatin in transgenerational epigenetic silencing.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Histona Metiltransferases/genética , Histonas/metabolismo , Interferência de RNA , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Histona Metiltransferases/metabolismo , RNA Nuclear/genética , RNA Nuclear/metabolismo
11.
Biosci Trends ; 14(4): 255-262, 2020 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-32350160

RESUMO

Salmonella enterica serovar Typhimurium (Salmonella), a pathogenic bacterium, is a major cause of foodborne diseases worldwide. Salmonella injects multiple virulence factors, called effectors, into cells and causes multiple rearrangements of cellular biological reactions that are important for Salmonella proliferation and virulence. Previously, we reported that Salmonella infection causes loss of MTR4 and RRP6, which are nuclear RNA degradation factors, resulting in the stabilization and accumulation of unstable nuclear RNAs. This accumulation is important for the cellular defense for Salmonella infection. In this study, we examined a series of Salmonella mutant strains, most of which are strains with genes related to effectors translocated by T3SSs encoded on Salmonella pathogenic islands, SPI-1 and SPI-2, that have been depleted. Among 42 Salmonella mutants, 6 mutants' infections canceled loss of MTR4 and RRP6. Proliferation assay of Salmonella in the cell revealed that six mutants showed poor proliferation in the host cell, demonstrating that poor proliferation contributed to cancellation of MTR4 and RRP6 loss. This result indicates that certain events associated with Salmonella proliferation in host cells cause loss of MTR4 and RRP6.


Assuntos
Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Helicases/metabolismo , Intoxicação Alimentar por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Proteólise , RNA Bacteriano/metabolismo , RNA Nuclear/metabolismo , Salmonella typhimurium/genética , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Virulência/genética , Fatores de Virulência/metabolismo
12.
Nucleic Acids Res ; 48(12): 6943-6953, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463452

RESUMO

ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5'cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.


Assuntos
Proteínas Nucleares/genética , RNA Helicases/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Células HEK293 , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas Nucleares/química , Domínios Proteicos/genética , RNA Helicases/química , RNA Mensageiro/química , RNA Nuclear/química , RNA Nuclear/genética
13.
Mol Cell ; 78(5): 862-875.e8, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32348780

RESUMO

Nuclear RNA interference (RNAi) pathways work together with histone modifications to regulate gene expression and enact an adaptive response to transposable RNA elements. In the germline, nuclear RNAi can lead to trans-generational epigenetic inheritance (TEI) of gene silencing. We identified and characterized a family of nuclear Argonaute-interacting proteins (ENRIs) that control the strength and target specificity of nuclear RNAi in C. elegans, ensuring faithful inheritance of epigenetic memories. ENRI-1/2 prevent misloading of the nuclear Argonaute NRDE-3 with small RNAs that normally effect maternal piRNAs, which prevents precocious nuclear translocation of NRDE-3 in the early embryo. Additionally, they are negative regulators of nuclear RNAi triggered from exogenous sources. Loss of ENRI-3, an unstable protein expressed mostly in the male germline, misdirects the RNAi response to transposable elements and impairs TEI. The ENRIs determine the potency and specificity of nuclear RNAi responses by gating small RNAs into specific nuclear Argonautes.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Inativação Gênica/fisiologia , Animais , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Células Germinativas/metabolismo , Proteínas Nucleares/metabolismo , Interferência de RNA/fisiologia , RNA de Cadeia Dupla/metabolismo , RNA Nuclear/metabolismo , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética
15.
Cell Rep ; 30(7): 2387-2401.e5, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32075771

RESUMO

Degradation of transcripts in human nuclei is primarily facilitated by the RNA exosome. To obtain substrate specificity, the exosome is aided by adaptors; in the nucleoplasm, those adaptors are the nuclear exosome-targeting (NEXT) complex and the poly(A) (pA) exosome-targeting (PAXT) connection. How these adaptors guide exosome targeting remains enigmatic. Employing high-resolution 3' end sequencing, we demonstrate that NEXT substrates arise from heterogenous and predominantly pA- 3' ends often covering kilobase-wide genomic regions. In contrast, PAXT targets harbor well-defined pA+ 3' ends defined by canonical pA site use. Irrespective of this clear division, NEXT and PAXT act redundantly in two ways: (1) regional redundancy, where the majority of exosome-targeted transcription units produce NEXT- and PAXT-sensitive RNA isoforms, and (2) isoform redundancy, where the PAXT connection ensures fail-safe decay of post-transcriptionally polyadenylated NEXT targets. In conjunction, this provides a two-layered targeting mechanism for efficient nuclear sorting of the human transcriptome.


Assuntos
Exossomos/metabolismo , Isoformas de Proteínas/metabolismo , RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Humanos
16.
Cells ; 9(2)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085641

RESUMO

The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.


Assuntos
Histonas/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , RNA Nuclear/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Animais , Núcleo Celular/metabolismo , Genoma Humano , Heterocromatina/metabolismo , Histonas/química , Histonas/genética , Humanos , Masculino , Camundongos , Camundongos Knockout , Células NIH 3T3 , Proteínas com Motivo de Reconhecimento de RNA/química , Proteínas com Motivo de Reconhecimento de RNA/genética , Motivos de Ligação ao RNA , Transfecção
17.
Science ; 367(6477): 580-586, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31949099

RESUMO

N 6-methyladenosine (m6A) regulates stability and translation of messenger RNA (mRNA) in various biological processes. In this work, we show that knockout of the m6A writer Mettl3 or the nuclear reader Ythdc1 in mouse embryonic stem cells increases chromatin accessibility and activates transcription in an m6A-dependent manner. We found that METTL3 deposits m6A modifications on chromosome-associated regulatory RNAs (carRNAs), including promoter-associated RNAs, enhancer RNAs, and repeat RNAs. YTHDC1 facilitates the decay of a subset of these m6A-modified RNAs, especially elements of the long interspersed element-1 family, through the nuclear exosome targeting-mediated nuclear degradation. Reducing m6A methylation by METTL3 depletion or site-specific m6A demethylation of selected carRNAs elevates the levels of carRNAs and promotes open chromatin state and downstream transcription. Collectively, our results reveal that m6A on carRNAs can globally tune chromatin state and transcription.


Assuntos
Adenosina/análogos & derivados , Cromatina/metabolismo , Metiltransferases/metabolismo , RNA Nuclear/metabolismo , Transcrição Genética , Adenosina/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos , Metilação , Metiltransferases/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo
18.
Nucleic Acids Res ; 48(5): 2518-2530, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31950173

RESUMO

Recruitment of the human ribonucleolytic RNA exosome to nuclear polyadenylated (pA+) RNA is facilitated by the Poly(A) Tail eXosome Targeting (PAXT) connection. Besides its core dimer, formed by the exosome co-factor MTR4 and the ZFC3H1 protein, the PAXT connection remains poorly defined. By characterizing nuclear pA+-RNA bound proteomes as well as MTR4-ZFC3H1 containing complexes in conditions favoring PAXT assembly, we here uncover three additional proteins required for PAXT function: ZC3H3, RBM26 and RBM27 along with the known PAXT-associated protein, PABPN1. The zinc-finger protein ZC3H3 interacts directly with MTR4-ZFC3H1 and loss of any of the newly identified PAXT components results in the accumulation of PAXT substrates. Collectively, our results establish new factors involved in the turnover of nuclear pA+ RNA and suggest that these are limiting for PAXT activity.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Exossomos/metabolismo , Poli A/metabolismo , Estabilidade de RNA , RNA Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Ligação Proteica , Proteoma/metabolismo , Ribonucleoproteínas/metabolismo
19.
Methods Mol Biol ; 2062: 467-489, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31768991

RESUMO

We describe procedures to clone, express, and reconstitute an active human nuclear RNA exosome. Individual recombinant subunits are expressed from E. coli and successfully reconstituted into the nuclear complex, which contains the noncatalytic nine-subunit exosome core, the endoribonuclease and exoribonuclease DIS3, the distributive exoribonuclease EXOSC10, the cofactors C1D and MPP6, and the RNA helicase MTR4.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , RNA Nuclear/metabolismo , RNA/metabolismo , Núcleo Celular/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/metabolismo , Exorribonucleases/metabolismo , Humanos , Subunidades Proteicas/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/metabolismo
20.
Plant Physiol ; 182(1): 147-158, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31722974

RESUMO

In addition to transcriptional regulation, gene expression is further modulated through mRNA spatiotemporal distribution, by RNA movement between cells, and by RNA localization within cells. Here, we have adapted RNA fluorescence in situ hybridization (FISH) to explore RNA localization in Arabidopsis (Arabidopsis thaliana). We show that RNA FISH on sectioned material can be applied to investigate the tissue and subcellular localization of meristem and flower development genes, cell cycle transcripts, and plant long noncoding RNAs. We also developed double RNA FISH to dissect the coexpression of different mRNAs at the shoot apex and nuclear-cytoplasmic separation of cell cycle gene transcripts in dividing cells. By coupling RNA FISH with fluorescence immunocytochemistry, we further demonstrate that a gene's mRNA and protein may be simultaneously detected, for example revealing uniform distribution of PIN-FORMED1 (PIN1) mRNA and polar localization of PIN1 protein in the same cells. Therefore, our method enables the visualization of gene expression at both transcriptional and translational levels with subcellular spatial resolution, opening up the possibility of systematically tracking the dynamics of RNA molecules and their cognate proteins in plant cells.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flores/metabolismo , Hibridização in Situ Fluorescente/métodos , RNA Nuclear/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação da Expressão Gênica de Plantas/genética , Meristema/genética , Meristema/metabolismo , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , RNA Nuclear/genética
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