RESUMO
BACKGROUND: High-mobility group B1 (HMGB1) is both a DNA binding nuclear factor modulating transcription and a crucial cytokine that mediates the response to both infectious and noninfectious inflammation such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. HMGB1 has been proposed to control ribosome biogenesis, similar as the other members of a class of HMGB proteins. RESULTS: Here, we report that HMGB1 selectively promotes transcription of genes involved in the regulation of transcription, osteoclast differentiation and apoptotic process. Improved RNA immunoprecipitation by UV cross-linking and deep sequencing (iRIP-seq) experiment revealed that HMGB1 selectively bound to mRNAs functioning not only in signal transduction and gene expression, but also in axon guidance, focal adhesion, and extracellular matrix organization. Importantly, HMGB1-bound reads were strongly enriched in specific structured RNAs, including the domain II of 28S rRNA, H/ACA box snoRNAs including snoRNA63 and scaRNAs. RTL-P experiment showed that overexpression of HMGB1 led to a decreased methylation modification of 28S rRNA at position Am2388, Cm2409, and Gm2411. We further showed that HMGB1 overexpression increased ribosome RNA expression levels and enhanced protein synthesis. CONCLUSION: Taken together, our results support a model in which HMGB1 binds to multiple RNA species in human cancer cells, which could at least partially contribute to HMGB1-modulated rRNA modification, protein synthesis function of ribosomes, and differential gene expression including rRNA genes. These findings provide additional mechanistic clues to HMGB1 functions in cancers and cell differentiation.
Assuntos
Proteína HMGB1 , Metilação de RNA , Humanos , Células HeLa , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Metilação , RNA Ribossômico 28S/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Metilação de RNA/genéticaRESUMO
The DEAD-box helicase Dbp4 plays an essential role during the early assembly of the 40S ribosome, which is only poorly understood to date. By applying the yeast two-hybrid method and biochemical approaches, we discovered that Dbp4 interacts with the Efg1-Bud22 dimer. Both factors associate with early pre-90S particles and smaller complexes, each characterized by a high presence of the U14 snoRNA. A crosslink analysis of Bud22 revealed its contact to the U14 snoRNA and the 5' domain of the nascent 18S rRNA, close to its U14 snoRNA hybridization site. Moreover, depletion of Bud22 or Efg1 specifically affects U14 snoRNA association with pre-ribosomal complexes. Accordingly, we concluded that the role of the Efg1-Bud22 dimer is linked to the U14 snoRNA function on early 90S ribosome intermediates chaperoning the 5' domain of the nascent 18S rRNA. The successful rRNA folding of the 5' domain and the release of Efg1, Bud22, Dpb4, U14 snoRNA and associated snoRNP factors allows the subsequent recruitment of the Kre33-Bfr2-Enp2-Lcp5 module towards the 90S pre-ribosome.
Assuntos
Ribonucleoproteínas Nucleolares Pequenas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ribonucleoproteínas Nucleolares Pequenas/genética , Ribossomos/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
Small nucleolar RNAs (snoRNAs) are structured noncoding RNAs present in multiple copies within eukaryotic genomes. snoRNAs guide chemical modifications on their target RNA and regulate processes like ribosome assembly and splicing. Most human snoRNAs are embedded within host gene introns, the remainder being independently expressed from intergenic regions. We recently characterized the abundance of snoRNAs and their host gene across several healthy human tissues and found that the level of most snoRNAs does not correlate with that of their host gene, with the observation that snoRNAs embedded within the same host gene often differ drastically in abundance. To better understand the determinants of snoRNA expression, we trained machine learning models to predict whether snoRNAs are expressed or not in human tissues based on more than 30 collected features related to snoRNAs and their genomic context. By interpreting the models' predictions, we find that snoRNAs rely on conserved motifs, a stable global structure and terminal stem, and a transcribed locus to be expressed. We observe that these features explain well the varying abundance of snoRNAs embedded within the same host gene. By predicting the expression status of snoRNAs across several vertebrates, we notice that only one-third of all annotated snoRNAs are expressed per genome, as in humans. Our results suggest that ancestral snoRNAs disseminated within vertebrate genomes, sometimes leading to the development of new functions and a probable gain in fitness and thereby conserving features favorable to the expression of these few snoRNAs, the large remainder often degenerating into pseudogenes.
Assuntos
RNA Nucleolar Pequeno , Vertebrados , Animais , Humanos , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/química , Vertebrados/genética , Eucariotos/genética , RNA não Traduzido , Expressão GênicaRESUMO
Background: The progression and recurrence of pterygium mainly occur due to the abnormal proliferation and migration of stromal pterygium fibroblasts. This research explores the aberrant expression of small nucleolar RNA U3 (U3 snoRNA) in pterygium and elucidates the molecular mechanisms of U3 snoRNA in pterygium development. Methods: Primary human conjunctival fibroblasts (HCFs) and human pterygium fibroblasts (HPFs) were separated and cultured from fresh conjunctiva grafts and pterygium tissues. The PLKO.1 lentiviral system and CRISPR/Cas9 recombinant construct were, respectively, used to overexpress and silence U3 snoRNA in HPFs and HCFs for further specific phenotype analysis. RNA-seq and TMT-labeled quantitative protein mass spectrometry were utilized to evaluate the effect of U3 snoRNA on mRNA transcripts and protein synthesis. Results: Reduced U3 snoRNA in pterygium promotes HCF or HPF cells' proliferation, migration, and cell cycle but has no significant effect on apoptosis. U3 snoRNA modulates 18S rRNA synthesis through shearing precursor ribosomal RNA 47S rRNA at the 5' external transcribed spacer (5' ETS). Moreover, the altered U3 snoRNA causes mRNA and protein differential expression in HCF or HPF cells. Conclusions: The atypical U3 snoRNA regulates the translation of specific proteins to exert a suppressive function in pterygium through modulating the 18S rRNA synthesis. Here, we uncover a novel insight into U3 snoRNA biology in the development of pterygium.
Assuntos
Pterígio , RNA Nucleolar Pequeno , Sequência de Bases , Túnica Conjuntiva/anormalidades , Túnica Conjuntiva/metabolismo , Humanos , Pterígio/genética , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Ribossômico , RNA Ribossômico 18S/química , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismoRESUMO
Characterization of RNA-protein interaction is fundamental for understanding the metabolism and function of RNA. UV crosslinking has been widely used to map the targets of RNA-binding proteins, but is limited by low efficiency, requirement for zero-distance contact, and biases for single-stranded RNA structure and certain residues of RNA and protein. Here, we report the development of an RNA-protein crosslinker (AMT-NHS) composed of a psoralen derivative and an N-hydroxysuccinimide ester group, which react with RNA bases and primary amines of protein, respectively. We show that AMT-NHS can penetrate into living yeast cells and crosslink Cbf5 to H/ACA snoRNAs with high specificity. The crosslinker induced different crosslinking patterns than UV and targeted both single- and double-stranded regions of RNA. The crosslinker provides a new tool to capture diverse RNA-protein interactions in cells.
Assuntos
Reagentes de Ligações Cruzadas/síntese química , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ficusina/química , Ligação Proteica , RNA Nucleolar Pequeno/química , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiaeRESUMO
The human small subunit processome mediates early maturation of the small ribosomal subunit by coupling RNA folding to subsequent RNA cleavage and processing steps. We report the high-resolution cryoelectron microscopy structures of maturing human small subunit (SSU) processomes at resolutions of 2.7 to 3.9 angstroms. These structures reveal the molecular mechanisms that enable crucial progressions during SSU processome maturation. RNA folding states within these particles are communicated to and coordinated with key enzymes that drive irreversible steps such as targeted exosome-mediated RNA degradation, protein-guided site-specific endonucleolytic RNA cleavage, and tightly controlled RNA unwinding. These conserved mechanisms highlight the SSU processome's impressive structural plasticity, which endows this 4.5-megadalton nucleolar assembly with the distinctive ability to mature the small ribosomal subunit from within.
Assuntos
Nucléolo Celular/ultraestrutura , Dobramento de RNA , Estabilidade de RNA , RNA Nucleolar Pequeno/química , Nucléolo Celular/metabolismo , Microscopia Crioeletrônica , RNA Helicases DEAD-box/química , Humanos , Clivagem do RNA , Fatores de Processamento de RNA/químicaRESUMO
In eukaryotes, rRNAs and spliceosomal snRNAs are heavily modified post-transcriptionally. Pseudouridylation and 2'-O-methylation are the most abundant types of RNA modifications. They are mediated by modification guide RNAs, also known as small nucleolar (sno)RNAs and small Cajal body-specific (sca)RNAs. We used yeast and vertebrate cells to test guide activities predicted for a number of snoRNAs, based on their regions of complementarity with rRNAs. We showed that human SNORA24 is a genuine guide RNA for 18S-Ψ609, despite some noncanonical base-pairing with its target. At the same time, we found quite a few snoRNAs that have the ability to base-pair with rRNAs and can induce predicted modifications in artificial substrate RNAs, but do not modify the same target sequence within endogenous rRNA molecules. Furthermore, certain fragments of rRNAs can be modified by the endogenous yeast modification machinery when inserted into an artificial backbone RNA, even though the same sequences are not modified in endogenous yeast rRNAs. In Xenopus cells, a guide RNA generated from scaRNA, but not from snoRNA, could induce an additional pseudouridylation of U2 snRNA at position 60; both guide RNAs were equally active on a U2 snRNA-specific substrate in yeast cells. Thus, post-transcriptional modification of functionally important RNAs, such as rRNAs and snRNAs, is highly regulated and more complex than simply strong base-pairing between a guide RNA and substrate RNA. We discuss possible regulatory roles for these unexpected modifications.
Assuntos
Corpos Enovelados/metabolismo , Pseudouridina/metabolismo , Processamento Pós-Transcricional do RNA , RNA Guia de Cinetoplastídeos/metabolismo , RNA Nucleolar Pequeno/metabolismo , Spliceossomos/metabolismo , Sequência de Bases , Corpos Enovelados/genética , Humanos , Metilação , RNA Guia de Cinetoplastídeos/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , Spliceossomos/genéticaRESUMO
Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3' UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.
Assuntos
RNA Fúngico/química , RNA não Traduzido/química , Regiões 3' não Traduzidas , Biologia Computacional/métodos , Genoma Fúngico , Íntrons , Lisina-tRNA Ligase/genética , Cadeias de Markov , Conformação de Ácido Nucleico , RNA Nucleolar Pequeno/química , Proteínas Ribossômicas/genética , Riboswitch , Alinhamento de Sequência , Tiorredoxinas/genéticaRESUMO
Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (-) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.
Assuntos
Doenças Transmissíveis/genética , Doenças Transmissíveis/metabolismo , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Fotoquímica/métodos , RNA/química , RNA/metabolismo , Calcinose/genética , Calcinose/metabolismo , Cistos do Sistema Nervoso Central/genética , Cistos do Sistema Nervoso Central/metabolismo , Reagentes de Ligações Cruzadas , Enterovirus Humano D/genética , Furocumarinas , Genoma Viral , Humanos , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Processos Fotoquímicos , RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA Viral/química , RNA Viral/genéticaRESUMO
The U3 promoter region of the HIV-1 long terminal repeat (LTR) has previously been shown to fold into a series of dynamic G-quadruplex structures. Among the G-quadruplexes identified in the LTR sequence, LTR-III was shown to be the most stable in vitro. NMR studies of this 28-nucleotide (nt) DNA revealed a unique quadruplex-hairpin structure. Whether the hairpin forms in RNA element is unknown and the role of the hairpin in the structure and stability of quadruplexes has not been characterized. Here, we used optical and thermodynamic studies to address these questions. The wild-type LTR-III RNA formed a monomolecular quadruplex with a parallel topology using only propeller loops, including the hairpin loop element. By comparison to the WT and variant RNAs, LTR-III DNA structures were more heterogeneous and less stable. Increased stability of the RNA suggests that the RNA quadruplex-hairpin structure may be a more attractive therapeutic target than the analogous DNA element.
Assuntos
HIV-1/genética , RNA Nucleolar Pequeno/química , RNA Viral/química , Quadruplex G , Conformação de Ácido Nucleico , RNA Nucleolar Pequeno/genética , RNA Viral/genéticaRESUMO
The presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multibasic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A/genética , RNA Nucleolar Pequeno/genética , RNA Viral/genética , Recombinação Genética , Aminoácidos Básicos/genética , Aminoácidos Básicos/metabolismo , Animais , Pareamento de Bases , Sequência de Bases , Galinhas/virologia , Códon , Regulação da Expressão Gênica , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Influenza Aviária/virologia , Influenza Humana/virologia , Mutagênese Insercional , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Alinhamento de Sequência , VirulênciaRESUMO
RNA helicases contribute to diverse aspects of RNA metabolism through their functions in re-arranging RNA structures. Identification of the remodelling targets of RNA helicases is a critical step in elucidating their cellular functions. Here, we show that, in contrast to many other ribosome biogenesis factors, the DExD box ATPase DDX55 predominantly localizes to the nucleoplasm and we identify a nuclear localization signal within the C-terminal region of the protein. DDX55 associates with pre-ribosomal subunits in human cells and is required for maturation of large subunit pre-rRNAs. Interestingly, in vitro analyses show that DDX55 selectively associates with double-stranded RNA substrates, which also stimulate its ATPase activity, and our data suggest that the C-terminal region of DDX55 contributes to this substrate specificity. The C-terminal region of DDX55 is also necessary for recruitment of the helicase to pre-ribosomes and, using in vivo crosslinking, we reveal a binding site for DDX55 in helix H62 of the 28S ribosomal RNA. Taken together, these data highlight the importance of the C-terminal region of DDX55 in substrate specificity and recruitment, and identify domain IV as a potential remodelling target of DDX55 during LSU biogenesis.
Assuntos
RNA Helicases DEAD-box/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico 28S/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , Células HEK293 , Células HeLa , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Biogênese de Organelas , Ligação Proteica , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Precursores de RNA/química , Precursores de RNA/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Ribossômico 28S/química , RNA Ribossômico 28S/genética , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribossomos/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
BACKGROUND: The discovery that much of the non-protein-coding genome is transcribed and plays a diverse functional role in fundamental cellular processes has led to an explosion in the development of tools and technologies to investigate the role of these noncoding RNAs in cardiovascular health. Furthermore, identifying noncoding RNAs for targeted therapeutics to treat cardiovascular disease is an emerging area of research. The purpose of this statement is to review existing literature, offer guidance on tools and technologies currently available to study noncoding RNAs, and identify areas of unmet need. METHODS: The writing group used systematic literature reviews (including MEDLINE, Web of Science through 2018), expert opinion/statements, analyses of databases and computational tools/algorithms, and review of current clinical trials to provide a broad consensus on the current state of the art in noncoding RNA in cardiovascular disease. RESULTS: Significant progress has been made since the initial studies focusing on the role of miRNAs (microRNAs) in cardiovascular development and disease. Notably, recent progress on understanding the role of novel types of noncoding small RNAs such as snoRNAs (small nucleolar RNAs), tRNA (transfer RNA) fragments, and Y-RNAs in cellular processes has revealed a noncanonical function for many of these molecules. Similarly, the identification of long noncoding RNAs that appear to play an important role in cardiovascular disease processes, coupled with the development of tools to characterize their interacting partners, has led to significant mechanistic insight. Finally, recent work has characterized the unique role of extracellular RNAs in mediating intercellular communication and their potential role as biomarkers. CONCLUSIONS: The rapid expansion of tools and pipelines for isolating, measuring, and annotating these entities suggests that caution in interpreting results is warranted until these methodologies are rigorously validated. Most investigators have focused on investigating the functional role of single RNA entities, but studies suggest complex interaction between different RNA molecules. The use of network approaches and advanced computational tools to understand the interaction of different noncoding RNA species to mediate a particular phenotype may be required to fully comprehend the function of noncoding RNAs in mediating disease phenotypes.
Assuntos
Doenças Cardiovasculares/genética , RNA não Traduzido/metabolismo , American Heart Association , Biomarcadores/metabolismo , Doenças Cardiovasculares/patologia , Humanos , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA não Traduzido/química , RNA não Traduzido/genética , Estados UnidosRESUMO
BACKGROUND: Increasing evidence supports the role of small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs) as master gene regulators at the epigenetic modification level. However, the underlying mechanism of these functional ncRNAs in colorectal cancer (CRC) has not been well investigated. METHODS: The dysregulated expression profiling of lncRNAs-snoRNAs-mRNAs and their correlations and co-expression enrichment were assessed by GeneChip microarray analysis. The candidate lncRNAs, snoRNAs, and target genes were detected by in situ hybridization (ISH), RT-PCR, qPCR and immunofluorescence (IF) assays. The biological functions of these factors were investigated using in vitro and in vivo studies that included CCK8, trans-well, cell apoptosis, IF assay, western blot method, and the xenograft mice models. rRNA 2'-O-methylation (Me) activities were determined by the RTL-P assay and a novel double-stranded primer based on the single-stranded toehold (DPBST) assay. The underlying molecular mechanisms were explored by bioinformatics and RNA stability, RNA fluorescence ISH, RNA pull-down and translation inhibition assays. RESULTS: To demonstrate the involvement of lncRNA and snoRNAs in 2'-O-Me modification during tumorigenesis, we uncovered a previously unreported mechanism linking the snoRNPs NOP58 regulated by ZFAS1 in control of SNORD12C, SNORD78 mediated rRNA 2'-O-Me activities in CRC initiation and development. Specifically, ZFAS1 exerts its oncogenic functions and significantly up-regulated accompanied by elevated NOP58, SNORD12C/78 expression in CRC cells and tissues. ZFAS1 knockdown suppressed CRC cell proliferation, migration, and increased cell apoptosis, and this inhibitory effect could be reversed by NOP58 overexpression in vitro and in vivo. Mechanistically, the NOP58 protein could be recognized by the specific motif (AAGA or CAGA) of ZFAS1. This event accelerates the assembly of SNORD12C/78 to allow for further guiding of 2'-O-Me at the corresponding Gm3878 and Gm4593 sites. Importantly, silencing SNORD12C or 78 reduced the rRNAs 2'-O-Me activities, which could be rescued by overexpression ZFAS1, and this subsequently inhibits the RNA stability and translation activity of their downstream targets (e.g., EIF4A3 and LAMC2). CONCLUSION: The novel ZFAS1-NOP58-SNORD12C/78-EIF4A3/LAMC2 signaling axis that functions in CRC tumorigenesis provides a better understanding regarding the role of lncRNA-snoRNP-mediated rRNAs 2'-O-Me activities for the prevention and treatment of CRC.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Metilação de DNA , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Nucleares/genética , Estabilidade de RNA , RNA Nucleolar Pequeno/química , Ribonucleoproteínas Nucleolares Pequenas/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Box C/D RNA protein complexes (RNPs) catalyze site-specific 2'-O-methylation of RNA with specificity determined by guide RNAs. In eukaryotic C/D RNP, the paralogous Nop58 and Nop56 proteins specifically associate with terminal C/D and internal C'/D' motifs of guide RNAs, respectively. We have reconstituted active C/D RNPs with recombinant proteins of the thermophilic yeast Chaetomium thermophilum. Nop58 and Nop56 could not distinguish between the two C/D motifs in the reconstituted enzyme, suggesting that the assembly specificity is imposed by trans-acting factors in vivo. The two C/D motifs are functionally independent and halfmer C/D RNAs can also guide site-specific methylation. Extensive pairing between C/D RNA and substrate is inhibitory to modification for both yeast and archaeal C/D RNPs. N6-methylated adenine at box D/D' interferes with the function of the coupled guide. Our data show that all C/D RNPs share the same functional organization and mechanism of action and provide insight into the assembly specificity of eukaryotic C/D RNPs.
Assuntos
Metiltransferases/química , Metiltransferases/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas/metabolismo , Adenina/análogos & derivados , Chaetomium/genética , Humanos , Metilação , Conformação de Ácido Nucleico , RNA/metabolismo , Ribonucleoproteínas/química , Sulfolobus solfataricusRESUMO
Longevity is influenced by genetic and environmental factors, but the underlying mechanisms remain elusive. Here, we functionally characterise a Drosophila small nucleolar RNA (snoRNA), named jouvence whose loss of function reduces lifespan. The genomic region of jouvence rescues the longevity in mutant, while its overexpression in wild-type increases lifespan. Jouvence is required in enterocytes. In mutant, the epithelium of the gut presents more hyperplasia, while the overexpression of jouvence prevents it. Molecularly, the mutant lack pseudouridylation on 18S and 28S-rRNA, a function rescued by targeted expression of jouvence in the gut. A transcriptomic analysis performed from the gut reveals that several genes are either up- or down-regulated, while restoring the mRNA level of two genes (ninaD or CG6296) rescue the longevity. Since snoRNAs are structurally and functionally well conserved throughout evolution, we identified putative jouvence orthologue in mammals including humans, suggesting that its function in longevity could be conserved.
Assuntos
Drosophila melanogaster/genética , Longevidade/genética , RNA Nucleolar Pequeno/genética , Animais , Animais Geneticamente Modificados , Sequência Conservada , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Enterócitos/metabolismo , Evolução Molecular , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Genoma de Inseto , Humanos , Mucosa Intestinal/metabolismo , Mutação com Perda de Função , Masculino , Camundongos , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Receptores Imunológicos/genéticaRESUMO
U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.
Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , Ribonucleoproteínas Nucleolares Pequenas/química , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Small nucleolar RNAs (snoRNAs) are an abundant type of non-coding RNA with conserved functions in all known eukaryotes. Classified into two main families, the box C/D and H/ACA snoRNAs, they enact their most well characterized role of guiding site specific modifications in ribosomal RNA, through the formation of specific ribonucleoprotein complexes, with fundamental implications in ribosome biogenesis. However, it is becoming increasingly clear that the landscape of snoRNA cellular functionality is much broader than it once seemed with novel members, non-uniform expression patterns, new and diverse targets as well as several emerging non-canonical functions ranging from the modulation of alternative splicing to the regulation of chromatin architecture. In order to facilitate the further characterization of human snoRNAs in a holistic manner, we introduce an online interactive database tool: snoDB. Its purpose is to consolidate information on human snoRNAs from different sources such as sequence databases, target information, both canonical and non-canonical from the literature and from high-throughput RNA-RNA interaction datasets, as well as high-throughput sequencing data that can be visualized interactively.
Assuntos
Bases de Dados de Ácidos Nucleicos , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de RNA , Interface Usuário-ComputadorRESUMO
Small nucleolar RNAs (snoRNAs) are short non-protein-coding RNAs with a long-recognized role in tuning ribosomal and spliceosomal function by guiding ribose methylation and pseudouridylation at targeted nucleotide residues of ribosomal and small nuclear RNAs, respectively. SnoRNAs are increasingly being implicated in regulation of new types of post-transcriptional processes, for example rRNA acetylation, modulation of splicing patterns, control of mRNA abundance and translational efficiency, or they themselves are processed to shorter stable RNA species that seem to be the principal or alternative bioactive isoform. Intriguingly, some display unusual cellular localization under exogenous stimuli, or tissue-specific distribution. Here, we discuss the new and unforeseen roles attributed to snoRNAs, focusing on the presumed mechanisms of action. Furthermore, we review the experimental approaches to study snoRNA function, including high resolution RNA:protein and RNA:RNA interaction mapping, techniques for analyzing modifications on targeted RNAs, and cellular and animal models used in snoRNA biology research.
Assuntos
Processamento de Proteína Pós-Traducional/genética , RNA Nucleolar Pequeno/genética , Ribossomos/genética , Spliceossomos/genética , Conformação de Ácido Nucleico , Splicing de RNA/genética , RNA Nucleolar Pequeno/química , Ribose/química , Ribose/genética , Uridina Monofosfato/metabolismoRESUMO
Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment. This structural motif has an internal three-nucleotide bulge flanked on its 3' side by a Gâ¢A/Aâ¢G tandem pairs followed by one or two non-Watson-Crick pairs, and on its 5' side by a classical RNA helix. This peculiar arrangement induces a strong curvature of the phosphodiester backbone, which makes it conducive to multiple tertiary interactions. SNU13/Snu13p (Human/Yeast) binds specifically the U14 C/D box snoRNA K-turn sequence motif. This event is the prerequisite to promote the assembly of the RNP, which contains NOP58/Nop58 and NOP56/Nop56 core proteins and the 2'-O-methyl-transferase, Fibrillarin/Nop1p. The U14 small nucleolar RNA is a conserved non-coding RNA found in yeast and vertebrates required for the pre-rRNA maturation and ribose methylation. Here, we report the solution structure of the free U14 snoRNA K-turn motif (kt-U14) as determined by Nuclear Magnetic Resonance. We demonstrate that a major fraction of free kt-U14 adopts a pre-folded conformation similar to protein bound K-turn, even in the absence of divalent ions. In contrast to the kt-U4 or tyrS RNA, kt-U14 displays a sharp bent in the phosphodiester backbone. The Uâ¢U and Gâ¢A tandem base pairs are formed through weak hydrogen bonds. Finally, we show that the structure of kt-U14 is stabilized upon Snu13p binding. The structure of the free U14 RNA is the first reference example for the canonical motifs of the C/D box snoRNA family.