RESUMO
In addition to phosphodiester bond formation, RNA polymerase II has an RNA endonuclease activity, stimulated by TFIIS, which rescues complexes that have arrested and backtracked. How TFIIS affects transcription under normal conditions is poorly understood. We identified backtracking sites in human cells using a dominant-negative TFIIS (TFIISDN) that inhibits RNA cleavage and stabilizes backtracked complexes. Backtracking is most frequent within 2 kb of start sites, consistent with slow elongation early in transcription, and in 3' flanking regions where termination is enhanced by TFIISDN, suggesting that backtracked pol II is a favorable substrate for termination. Rescue from backtracking by RNA cleavage also promotes escape from 5' pause sites, prevents premature termination of long transcripts, and enhances activation of stress-inducible genes. TFIISDN slowed elongation rates genome-wide by half, suggesting that rescue of backtracked pol II by TFIIS is a major stimulus of elongation under normal conditions.
Assuntos
Clivagem do RNA , RNA Polimerase II/metabolismo , RNA/metabolismo , Elongação da Transcrição Genética , Terminação da Transcrição Genética , Ativação Transcricional , Região 3'-Flanqueadora , Animais , Regulação da Expressão Gênica , Células HEK293 , Humanos , Cinética , Camundongos , Mutação , RNA/genética , RNA Polimerase II/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
Dosage compensation, mediated by the MSL complex, regulates X-chromosomal gene expression in Drosophila. Here we report that the histone H4 lysine 16 (H4K16) specific histone acetyltransferase MOF displays differential binding behavior depending on whether the target gene is located on the X chromosome versus the autosomes. More specifically, on the male X chromosome, where MSL1 and MSL3 are preferentially associated with the 3' end of dosage compensated genes, MOF displays a bimodal distribution binding to promoters and the 3' ends of genes. In contrast, on MSL1/MSL3 independent X-linked genes and autosomal genes in males and females, MOF binds primarily to promoters. Binding of MOF to autosomes is functional, as H4K16 acetylation and the transcription levels of a number of genes are affected upon MOF depletion. Therefore, MOF is not only involved in the onset of dosage compensation, but also acts as a regulator of gene expression in the Drosophila genome.
Assuntos
Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Proteínas Nucleares/metabolismo , Região 3'-Flanqueadora , Acetilação , Animais , Linhagem Celular , Feminino , Genoma de Inseto , Histonas/genética , Histonas/metabolismo , Masculino , Regiões Promotoras Genéticas , Cromossomo XRESUMO
Cyclobutane pyrimidine dimers (CPDs) are the major products of DNA produced by direct absorption of UV light, and result in C to T mutations linked to human skin cancers. Most recently a new pathway to CPDs in melanocytes has been discovered that has been proposed to arise from a chemisensitized pathway involving a triplet sensitizer that increases mutagenesis by increasing the percentage of C-containing CPDs. To investigate how triplet sensitization may differ from direct UV irradiation, CPD formation was quantified in a 129-mer DNA designed to contain all 64 possible NYYN sequences. CPD formation with UVB light varied about 2-fold between dipyrimidines and 12-fold with flanking sequence and was most frequent at YYYR and least frequent for GYYN sites in accord with a charge transfer quenching mechanism. In contrast, photosensitized CPD formation greatly favored TT over C-containing sites, more so for norfloxacin (NFX) than acetone, in accord with their differing triplet energies. While the sequence dependence for photosensitized TT CPD formation was similar to UVB light, there were significant differences, especially between NFX and acetone that could be largely explained by the ability of NFX to intercalate into DNA.
Assuntos
Região 3'-Flanqueadora , Região 5'-Flanqueadora , DNA/química , DNA/efeitos da radiação , Fármacos Fotossensibilizantes/química , Dímeros de Pirimidina/química , Sequência de Bases , Citosina/química , Humanos , Melanócitos/química , Melanócitos/efeitos da radiação , Mutagênese , Mutação , Neoplasias Cutâneas/genética , Timina/química , Raios UltravioletaRESUMO
Nibbler (Nbr) is a 3'-to-5' exoribonuclease whose catalytic 3'-end trimming activity impacts microRNA (miRNA) and PIWI-interacting RNA (piRNA) biogenesis. Here, we report on structural and functional studies to decipher the contributions of Nbr's N-terminal domain (NTD) and exonucleolytic domain (EXO) in miRNA 3'-end trimming. We have solved the crystal structures of the NTD core and EXO domains of Nbr, both in the apo-state. The NTD-core domain of Aedes aegypti Nbr adopts a HEAT-like repeat scaffold with basic patches constituting an RNA-binding surface exhibiting a preference for binding double-strand RNA (dsRNA) over single-strand RNA (ssRNA). Structure-guided functional assays in Drosophila S2 cells confirmed a principal role of the NTD in exonucleolytic miRNA trimming, which depends on basic surface patches. Gain-of-function experiments revealed a potential role of the NTD in recruiting Nbr to Argonaute-bound small RNA substrates. The EXO domain of A. aegypti and Drosophila melanogaster Nbr adopt a mixed α/ß-scaffold with a deep pocket lined by a DEDDy catalytic cleavage motif. We demonstrate that Nbr's EXO domain exhibits Mn2+-dependent ssRNA-specific 3'-to-5' exoribonuclease activity. Modeling of a 3' terminal Uridine into the catalytic pocket of Nbr EXO indicates that 2'-O-methylation of the 3'-U would result in a steric clash with a tryptophan side chain, suggesting that 2'-O-methylation protects small RNAs from Nbr-mediated trimming. Overall, our data establish that Nbr requires its NTD as a substrate recruitment platform to execute exonucleolytic miRNA maturation, catalyzed by the ribonuclease EXO domain.
Assuntos
Região 3'-Flanqueadora , Proteínas de Drosophila/química , Exorribonucleases/química , MicroRNAs/química , MicroRNAs/genética , Processamento Pós-Transcricional do RNA , Relação Estrutura-Atividade , Animais , Proteínas Argonautas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Exorribonucleases/metabolismo , MicroRNAs/metabolismo , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismoRESUMO
Short interspersed nuclear elements (SINEs) are small, non-autonomous and heterogeneous retrotransposons that are widespread in plants. To explore the amplification dynamics and evolutionary history of SINE populations in representative deciduous tree species, we analyzed the genomes of the six following Salicaceae species: Populus deltoides, Populus euphratica, Populus tremula, Populus tremuloides, Populus trichocarpa, and Salix purpurea. We identified 11 Salicaceae SINE families (SaliS-I to SaliS-XI), comprising 27 077 full-length copies. Most of these families harbor segmental similarities, providing evidence for SINE emergence by reshuffling or heterodimerization. We observed two SINE groups, differing in phylogenetic distribution pattern, similarity and 3' end structure. These groups probably emerged during the 'salicoid duplication' (~65 million years ago) in the Salix-Populus progenitor and during the separation of the genus Salix (45-65 million years ago), respectively. In contrast to conserved 5' start motifs across species and SINE families, the 3' ends are highly variable in sequence and length. This extraordinary 3'-end variability results from mutations in the poly(A) tail, which were fixed by subsequent amplificational bursts. We show that the dissemination of newly evolved 3' ends is accomplished by a displacement of older motifs, leading to various 3'-end subpopulations within the SaliS families.
Assuntos
Região 3'-Flanqueadora/genética , Salicaceae/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Evolução Biológica , Mapeamento Cromossômico , Cromossomos de Plantas/genética , Sequência Conservada/genética , Genes de Plantas/genética , Genoma de Planta/genética , Filogenia , Populus/genética , Salix/genéticaRESUMO
Characteristically, cancer cells metabolize glucose through aerobic glycolysis, known as the Warburg effect. Accumulating evidence suggest that during cancer formation, microRNAs (miRNAs) could regulate such metabolic reprogramming. In the present study, miR-9-1 was identified as significantly hypermethylated in nasopharyngeal carcinoma (NPC) cell lines and clinical tissues. Ectopic expression of miR-9-1 inhibited NPC cell growth and glycolytic metabolism, including reduced glycolysis, by reducing lactate production, glucose uptake, cellular glucose-6-phosphate levels, and ATP generation in vitro and tumor proliferation in vivo. HK2 (encoding hexokinase 2) was identified as a direct target of miR-9-1 using luciferase reporter assays and Western blotting. In NPC cells, hypermethylation regulates miR-9-1 expression and inhibits HK2 translation by directly targeting its 3' untranslated region. MiR-9-1 overexpression markedly reduced HK2 protein levels. Restoration of HK2 expression attenuated the inhibitory effect of miR-9-1 on NPC cell proliferation and glycolysis. Fluorescence in situ hybridization results indicated that miR-9-1 expression was an independent prognostic factor in NPC. Our findings revealed the role of the miR-9-1/HK2 axis in the metabolic reprogramming of NPC, providing a potential therapeutic strategy for NPC.
Assuntos
Hexoquinase/metabolismo , MicroRNAs/metabolismo , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Região 3'-Flanqueadora , Trifosfato de Adenosina/biossíntese , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Glucose/metabolismo , Glucose-6-Fosfato/metabolismo , Glicólise , Xenoenxertos , Humanos , Hibridização in Situ Fluorescente , Ácido Láctico/biossíntese , Masculino , Metilação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Carcinoma Nasofaríngeo/mortalidade , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/mortalidade , Neoplasias Nasofaríngeas/patologia , Células-Tronco Neoplásicas , RNA Mensageiro/metabolismoRESUMO
Thousands of eukaryotic protein-coding genes generate circular RNAs that have covalently linked ends and are resistant to degradation by exonucleases. To prove their circularity as well as biochemically enrich these transcripts, it has become standard in the field to use the 3'-5' exonuclease RNase R. Here, we demonstrate that standard protocols involving RNase R can fail to digest >20% of all highly expressed linear RNAs, but these shortcomings can largely be overcome. RNAs with highly structured 3' ends, including snRNAs and histone mRNAs, are naturally resistant to RNase R, but can be efficiently degraded once a poly(A) tail has been added to their ends. In addition, RNase R stalls in the body of many polyadenylated mRNAs, especially at G-rich sequences that have been previously annotated as G-quadruplex (G4) structures. Upon replacing K+ (which stabilizes G4s) with Li+ in the reaction buffer, we find that RNase R is now able to proceed through these sequences and fully degrade the mRNAs in their entirety. In total, our results provide important improvements to the current methods used to isolate circular RNAs as well as a way to reveal RNA structures that may naturally inhibit degradation by cellular exonucleases.
Assuntos
Exorribonucleases/química , Quadruplex G , RNA Mensageiro/isolamento & purificação , RNA Nuclear Pequeno/genética , RNA/isolamento & purificação , Região 3'-Flanqueadora , Células HeLa , Humanos , Lítio/farmacologia , Poliadenilação , Potássio/farmacologia , RNA/química , RNA/genética , RNA/metabolismo , Clivagem do RNA/efeitos dos fármacos , RNA Circular , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Análise de Sequência de RNARESUMO
The t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics. Inhibition of CDK7 using THZ1 caused wide-spread loss of promoter-proximal paused RNA polymerase. This loss of 5' pausing was associated with accumulation of polymerases in the body of a large number of genes. However, there were modest effects on genes regulated by 'super-enhancers'. At the 3' ends of genes, treatment with THZ1 suppressed RNA polymerase 'read through' at the end of the last exon, which resembled a phenotype associated with a mutant RNA polymerase with slower elongation rates. Consistent with this hypothesis, polyA site-sequencing (PolyA-seq) did not detect differences in poly A sites after THZ1 treatment. PROseq analysis after short treatments with THZ1 suggested that these 3' effects were due to altered CDK7 activity at the 5' end of long genes, and were likely to be due to slower rates of elongation.
Assuntos
Antineoplásicos/farmacologia , Quinases Ciclina-Dependentes/genética , Regulação Leucêmica da Expressão Gênica , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Polimerase II/genética , Região 3'-Flanqueadora , Região 5'-Flanqueadora/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Óxidos N-Cíclicos , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Flavonoides/farmacologia , Humanos , Indolizinas , Células Mieloides/metabolismo , Células Mieloides/patologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Piperidonas/farmacologia , Piridinas/farmacologia , Compostos de Piridínio/farmacologia , Pirróis/farmacologia , RNA Polimerase II/antagonistas & inibidores , RNA Polimerase II/metabolismo , Translocação Genética , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
Transcription is a major obstacle for replication fork (RF) progression and a cause of genome instability. Part of this instability is mediated by cotranscriptional R loops, which are believed to increase by suboptimal assembly of the nascent messenger ribonucleoprotein particle (mRNP). However, no clear evidence exists that heterogeneous nuclear RNPs (hnRNPs), the basic mRNP components, prevent R-loop stabilization. Here we show that yeast Npl3, the most abundant RNA-binding hnRNP, prevents R-loop-mediated genome instability. npl3Δ cells show transcription-dependent and R-loop-dependent hyperrecombination and genome-wide replication obstacles as determined by accumulation of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Npl3 is preferentially bound in wild-type cells, and are reduced by RNase H1 overexpression. The resulting replication stress confers hypersensitivity to double-strand break-inducing agents. Therefore, our work demonstrates that mRNP factors are critical for genome integrity and opens the option of using them as therapeutic targets in anti-cancer treatment.
Assuntos
Replicação do DNA/genética , Instabilidade Genômica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/genética , Região 3'-Flanqueadora , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , Deleção de Genes , Genoma Fúngico , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Mutagênicos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacosRESUMO
We improved miR-143, which inhibits the growth of cancer cells, by the replacement of the passenger strand. As a result, new miR-143 variants were developed with a single mismatch at the 4th position from the 3'-terminal of the guide strand and an RNA passenger strand with a G-rich flanking DNA region. A reporter gene assay showed that the 80% inhibitory concentration of the new miR-143, long miR-143, was 69 pM, which was three times lower than that of natural miR-143. Long miR-143 inhibited the growth of two cancer cell lines, HeLa-S3 and MIAPaCa-2, more effectively than natural miR-143. This method could be applied to other miRNA families and should be useful for the development of miRNA drugs.
Assuntos
Regulação Neoplásica da Expressão Gênica , Inativação Gênica , MicroRNAs/genética , Oligonucleotídeos/genética , RNA Mensageiro/genética , Região 3'-Flanqueadora , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , MicroRNAs/metabolismo , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Variable-(diversity)-joining (V(D)J) recombination at loci encoding the immunoglobulin heavy chain (Igh) and immunoglobulin light chain (Igk) takes place sequentially during successive stages in B cell development. Using three-dimensional DNA fluorescence in situ hybridization, here we identify a lineage-specific and stage-specific interchromosomal association between these two loci that marks the transition between Igh and Igk recombination. Colocalization occurred between pericentromerically located alleles in pre-B cells and was mediated by the 3' Igk enhancer. Deletion of this regulatory element prevented association of the Igh and Igk loci, inhibited pericentromeric recruitment and locus 'decontraction' of an Igh allele, and resulted in greater distal rearrangement of the gene encoding the variable heavy-chain region. Our data indicate involvement of the Igk locus and its 3' enhancer in directing the Igh locus to a repressive nuclear subcompartment and inducing the Igh locus to decontract.
Assuntos
Elementos Facilitadores Genéticos/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Genes de Cadeia Pesada de Imunoglobulina , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulinas/genética , Células Precursoras de Linfócitos B/imunologia , Região 3'-Flanqueadora/genética , Animais , Cromossomos/genética , Cromossomos/metabolismo , Genes de Cadeia Pesada de Imunoglobulina/fisiologia , Imunoglobulinas/fisiologia , Camundongos , Camundongos Knockout , Células Precursoras de Linfócitos B/metabolismo , Recombinação GenéticaRESUMO
The post-transcriptional modification 2'-O-Methyl (2'OMe) could be present on the ribose of all four ribonucleosides, and is highly prevalent in a wide variety of RNA species, including the 5' RNA cap of viruses and higher eukaryotes, as well as internally in transfer RNA and ribosomal RNA. Recent studies have suggested that 2'OMe is also located internally in low-abundance RNA species such as viral RNA and mRNA. To profile 2'OMe on different RNA species, we have developed Nm-seq, which could identify 2'OMe sites at single base resolution. Nm-seq is particularly useful for identifying 2'OMe sites located at the 3' terminal ends of small RNAs. Here, we present an optimized protocol for Nm-seq and a protocol for applying Nm-seq to identify 2'OMe sites on small RNA 3' terminal ends.
Assuntos
MicroRNAs/genética , Anotação de Sequência Molecular/métodos , Poli A/genética , RNA Mensageiro/genética , Região 3'-Flanqueadora , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Biblioteca Gênica , Humanos , Hidrólise , Metilação , MicroRNAs/metabolismo , Oxirredução , Fosforilação , Poli A/metabolismo , RNA Mensageiro/metabolismo , Ribose/metabolismo , Plântula/genética , Plântula/metabolismoRESUMO
Post-transcriptional modification of RNA, the so-called 'Epitranscriptome', can regulate RNA structure, stability, localization, and function. Numerous modifications have been identified in virtually all classes of RNAs, including messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), microRNAs (miRNAs), and other noncoding RNAs (ncRNAs). These modifications may occur internally (by base or sugar modifications) and include RNA methylation at different nucleotide positions, or by the addition of various nucleotides at the 3'-end of certain transcripts by a family of terminal nucleotidylyl transferases. Developing methods to specifically and accurately detect and map these modifications is essential for understanding the molecular function(s) of individual RNA modifications and also for identifying and characterizing the proteins that may read, write, or erase them. Here, we focus on the characterization of RNA species targeted by 3' terminal uridylyl transferases (TUTases) (TUT4/7, also known as Zcchc11/6) and a 3'-5' exoribonuclease, Dis3l2, in the recently identified Dis3l2-mediated decay (DMD) pathway - a dedicated quality control pathway for a subset of ncRNAs. We describe the detailed methods used to precisely identify 3'-end modifications at nucleotide level resolution with a particular focus on the U1 and U2 small nuclear RNA (snRNA) components of the Spliceosome. These tools can be applied to investigate any RNA of interest and should facilitate studies aimed at elucidating the functional relevance of 3'-end modifications.
Assuntos
Biologia Computacional/métodos , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Nuclear Pequeno/genética , Uridina/metabolismo , Região 3'-Flanqueadora , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Exorribonucleases/deficiência , Exorribonucleases/genética , Edição de Genes/métodos , Camundongos , Células-Tronco Embrionárias Murinas , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estabilidade de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
The use of RNA-seq as a generalized tool to measure the differential expression of genes has essentially replaced the use of the microarray. Despite the acknowledged technical advantages to this approach, RNA-seq library preparation remains mostly conducted by core facilities rather than in the laboratory due to the infrastructure, expertise and time required per sample. We have recently described two 'click-chemistry' based library construction methods termed ClickSeq and Poly(A)-ClickSeq (PAC-seq) as alternatives to conventional RNA-seq that are both cost effective and rely on straightforward reagents readily available to most labs. ClickSeq is random-primed and can sequence any (unfragmented) RNA template, while PAC-seq is targeted to poly(A) tails of mRNAs. Here, we further develop PAC-seq as a platform that allows for simultaneous mapping of poly(A) sites and the measurement of differential expression of genes. We provide a detailed protocol, descriptions of appropriate computational pipelines, and a proof-of-principle dataset to illustrate the technique. PAC-seq offers a unique advantage over other 3' end mapping protocols in that it does not require additional purification, selection, or fragmentation steps allowing sample preparation directly from crude total cellular RNA. We have shown that PAC-seq is able to accurately and sensitively count transcripts for differential gene expression analysis, as well as identify alternative poly(A) sites and determine the precise nucleotides of the poly(A) tail boundaries.
Assuntos
Química Click/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Insetos/genética , Poli A/genética , RNA Mensageiro/genética , Região 3'-Flanqueadora , Animais , Células Cultivadas , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Biblioteca Gênica , Genoma de Inseto , Proteínas de Insetos/metabolismo , Poli A/química , Poli A/metabolismo , Poliadenilação , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/estatística & dados numéricosRESUMO
S-adenosyl-L-methionine-dependent 2'-O-methylati-on of the 3'-terminal nucleotide plays important roles in biogenesis of eukaryotic small non-coding RNAs, such as siRNAs, miRNAs and Piwi-interacting RNAs (piRNAs). Here we demonstrate that, in contrast to Mg2+/Mn2+-dependent plant and bacterial homologues, the Drosophila DmHen1 and human HsHEN1 piRNA methyltransferases require cobalt cations for their enzymatic activity in vitro. We also show for the first time the capacity of the animal Hen1 to catalyse the transfer of a variety of extended chemical groups from synthetic analogues of the AdoMet cofactor onto a wide range (22-80 nt) of single-stranded RNAs permitting their 3'-terminal functionalization and labelling. Moreover, we provide evidence that deletion of a small C-terminal region of the DmHen1 protein further increases its modification efficiency and abolishes a modest 3'-terminal nucleotide bias observed for the full-length protein. Finally, we show that fluorophore-tagged ssRNA molecules are successfully detected in fluorescence resonance energy transfer assays both individually and in a total RNA mixture. The presented DmHen1-assisted RNA labelling provides a solid basis for developing novel chemo-enzymatic approaches for in vitro studies and in vivo monitoring of single-stranded RNA pools.
Assuntos
Região 3'-Flanqueadora , Proteínas de Drosophila/fisiologia , Metiltransferases/fisiologia , RNA/metabolismo , Coloração e Rotulagem/métodos , Região 3'-Flanqueadora/genética , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células HCT116 , Humanos , Metiltransferases/metabolismo , MicroRNAs/metabolismo , RNA/química , Processamento de Terminações 3' de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Imagem Individual de Molécula/métodosRESUMO
A polymerase ribozyme can be used to label the 3' end of RNA or DNA molecules by incorporating a variety of functionalized nucleotide analogs. Guided by a complementary template, the ribozyme adds a single nucleotide that may contain a fluorophore, biotin, azide or alkyne moiety, thus enabling the detection and/or capture of selectively labeled materials. Employing a variety of commercially available nucleotide analogs, efficient labeling was demonstrated for model RNAs and DNAs, human microRNAs and natural tRNA.
Assuntos
Região 3'-Flanqueadora , RNA Polimerases Dirigidas por DNA/metabolismo , Ácidos Nucleicos/metabolismo , RNA Catalítico/metabolismo , Coloração e Rotulagem/métodos , Biotina/química , Biotina/metabolismo , DNA/química , DNA/metabolismo , Fluoresceína/química , Fluoresceína/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , MicroRNAs/química , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , Ácidos Nucleicos/química , Nucleotidiltransferases/metabolismo , RNA/química , RNA/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Rodaminas/química , Rodaminas/metabolismoRESUMO
ATP-dependent chromatin remodeling complexes are important regulators of gene expression in Eukaryotes. In plants, SWI/SNF-type complexes have been shown critical for transcriptional control of key developmental processes, growth and stress responses. To gain insight into mechanisms underlying these roles, we performed whole genome mapping of the SWI/SNF catalytic subunit BRM in Arabidopsis thaliana, combined with transcript profiling experiments. Our data show that BRM occupies thousands of sites in Arabidopsis genome, most of which located within or close to genes. Among identified direct BRM transcriptional targets almost equal numbers were up- and downregulated upon BRM depletion, suggesting that BRM can act as both activator and repressor of gene expression. Interestingly, in addition to genes showing canonical pattern of BRM enrichment near transcription start site, many other genes showed a transcription termination site-centred BRM occupancy profile. We found that BRM-bound 3Î gene regions have promoter-like features, including presence of TATA boxes and high H3K4me3 levels, and possess high antisense transcriptional activity which is subjected to both activation and repression by SWI/SNF complex. Our data suggest that binding to gene terminators and controlling transcription of non-coding RNAs is another way through which SWI/SNF complex regulates expression of its targets.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Regiões Promotoras Genéticas , Regiões Terminadoras Genéticas , Região 3'-Flanqueadora , Arabidopsis/metabolismo , Sítios de Ligação , RNA Antissenso/biossíntese , RNA Mensageiro/biossíntese , Transcrição GênicaRESUMO
Cells regulate biological responses in part through changes in transcription start sites (TSS) or cleavage and polyadenylation sites (PAS). To fully understand gene regulatory networks, it is therefore critical to accurately annotate cell type-specific TSS and PAS. Here we present a simple and straightforward approach for genome-wide annotation of 5Î- and 3Î-RNA ends. Our approach reliably discerns bona fide PAS from false PAS that arise due to internal poly(A) tracts, a common problem with current PAS annotation methods. We applied our methodology to study the impact of temperature on the Drosophila melanogaster head transcriptome. We found hundreds of previously unidentified TSS and PAS which revealed two interesting phenomena: first, genes with multiple PASs tend to harbor a motif near the most proximal PAS, which likely represents a new cleavage and polyadenylation signal. Second, motif analysis of promoters of genes affected by temperature suggested that boundary element association factor of 32 kDa (BEAF-32) and DREF mediates a transcriptional program at warm temperatures, a result we validated in a fly line where beaf-32 is downregulated. These results demonstrate the utility of a high-throughput platform for complete experimental and computational analysis of mRNA-ends to improve gene annotation.
Assuntos
Drosophila melanogaster/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Animais , Sequência de Bases , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Exonucleases/química , Genes de Insetos , Anotação de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease H/química , TranscriptomaRESUMO
The detailed analysis of the impact of deletions on proteins or nucleic acids can reveal important functional regions and lead to variants with improved macromolecular properties. We present a method to generate large libraries of mutants with deletions of varying length that are randomly distributed throughout a given gene. This technique facilitates the identification of crucial sequence regions in nucleic acids or proteins. The approach utilizes in vitro transposition to generate 5Î and 3Î fragment sub-libraries of a given gene, which are then randomly recombined to yield a final library comprising both terminal and internal deletions. The method is easy to implement and can generate libraries in three to four days. We used this approach to produce a library of >9000 random deletion mutants of an artificial RNA ligase enzyme representing 32% of all possible deletions. The quality of the library was assessed by next-generation sequencing and detailed bioinformatics analysis. Finally, we subjected this library to in vitro selection and obtained fully functional variants with deletions of up to 18 amino acids of the parental enzyme that had been 95 amino acids in length.
Assuntos
Sequência de Aminoácidos , DNA/genética , Biblioteca Gênica , Deleção de Sequência , Transposases/genética , Região 3'-Flanqueadora , Região 5'-Flanqueadora , Biologia Computacional , DNA/metabolismo , Primers do DNA/genética , Primers do DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transposases/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Chlamydomonas reinhardtii is a unicellular green alga that has attracted interest due to its potential biotechnological applications, and as a model for algal biofuel and energy metabolism. Despite all the advantages that this unicellular alga offers, poor and inconsistent expression of nuclear transgenes remains an obstacle for basic and applied research. We used a data-mining strategy to identify highly expressed genes in Chlamydomonas whose flanking sequences were tested for the ability to drive heterologous nuclear transgene expression. Candidates identified in this search included two ribosomal protein genes, RPL35a and RPL23, and ferredoxin, FDX1, whose flanking regions including promoters, terminators and untranslated sequences could drive stable luciferase transgene expression to significantly higher levels than the commonly used Hsp70A-RBCS2 (AR) hybrid promoter/terminator sequences. The RPL23 flanking sequences were further tested using the zeocin resistance gene sh-ble as a reporter in monocistronic and dicistronic constructs, and consistently yielded higher numbers of zeocin-resistant transformants and higher levels of resistance than AR- or PSAD-based vectors. Chlamydomonas RPL23 sequences also enabled transgene expression in Volvox carteri. Our study provides an additional benchmark for strong constitutive expression of transgenes in Chlamydomonas, and develops a general approach for identifying flanking sequences that can be used to drive transgene expression for any organism where transcriptome data are available.