RESUMO
Transcripts from the human WDR33 gene, which encodes a central component of the mRNA polyadenylation (PA) machinery, are subject to alternative polyadenylation (APA) within promoter-proximal introns/exons. This APA, which itself involves usage of multiple PA sites, results in the production of two non-canonical protein isoforms, V2 and V3, that are functionally completely unrelated to the full-length protein, with roles in innate immunity. The mechanism and regulation of WDR33 APA are unclear. Here, we report that levels of the PA factor CFIm25 modulate V2 and V3 expression, and that PA site usage of both V2 and V3 varies in distinct immune responses. Using newly developed assays to measure splicing and PA site strength, we show that splicing of V2-associated intron 6 is inefficient, allowing V2 to be produced using weak PA sites. Usage of V3's strong PA sites, on the other hand, is relatively low, reflecting the high efficiency of intron 7 splicing coupled with dependency on usage of an alternative 3' splice site within the intron. Overall, our findings demonstrate that usage of WDR33 alternative PA sites is stochastic, dependent on a complex interplay between splicing and PA, and thus provide new insights into mechanisms underlying APA.
Assuntos
Processamento Alternativo , Íntrons , Poliadenilação , Humanos , Poli A/metabolismo , Poli A/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Éxons , Splicing de RNA , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMO
Cytoplasmic mRNA decay is effected by exonucleolytic degradation in either the 5' to 3' or 3' to 5' direction. Pervasive terminal uridylation is implicated in mRNA degradation, however, its functional relevance for bulk mRNA turnover remains poorly understood. In this study, we employ genome-wide 3'-RACE (gw3'-RACE) in the model system fission yeast to elucidate the role of uridylation in mRNA turnover. We observe widespread uridylation of shortened poly(A) tails, promoting efficient 5' to 3' mRNA decay and ensuring timely and controlled mRNA degradation. Inhibition of this uridylation process leads to excessive deadenylation and enhanced 3' to 5' mRNA decay accompanied by oligouridylation. Strikingly we found that uridylation of poly(A) tails and oligouridylation of non-polyadenylated substrates are catalysed by different terminal uridyltransferases Cid1 and Cid16 respectively. Our study sheds new light on the intricate regulatory mechanisms underlying bulk mRNA turnover, demonstrating the role of uridylation in modulating mRNA decay pathways.
Assuntos
Estabilidade de RNA , RNA Mensageiro , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Poli A/metabolismo , Uridina/metabolismo , Regulação Fúngica da Expressão Gênica , RNA Fúngico/metabolismo , RNA Fúngico/genética , NucleotidiltransferasesRESUMO
Quantifying the number of proteins that interact with mRNAs, in particular with poly(A) tails of mRNAs, is crucial for understanding gene regulation. Biochemical assays offer significant advantages for this purpose. Here, we present a protocol for synthesizing mRNAs with accurate, length-specific poly(A) tails through a PCR-based approach. We also describe steps for an in vitro (i.e., cell-free) approach for visualizing the sequential binding of Cytoplasmic Poly(A)-Binding Proteins (PABPCs) to these poly(A) tails. We detail quality control steps throughout the procedure. For complete details on the use and execution of this protocol, please refer to Grandi et al.1.
Assuntos
Bioquímica , Poli A , Proteínas de Ligação a Poli(A) , RNA Mensageiro , Humanos , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/genética , Reação em Cadeia da Polimerase/métodos , Ligação Proteica , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Transcrição Gênica/genética , Bioquímica/métodosRESUMO
Oocyte in vitro maturation is a technique in assisted reproductive technology. Thousands of genes show abnormally high expression in in vitro maturated metaphase II (MII) oocytes compared to those matured in vivo in bovines, mice, and humans. The mechanisms underlying this phenomenon are poorly understood. Here, we use poly(A) inclusive RNA isoform sequencing (PAIso-seq) for profiling the transcriptome-wide poly(A) tails in both in vivo and in vitro matured mouse and human oocytes. Our results demonstrate that the observed increase in maternal mRNA abundance is caused by impaired deadenylation in in vitro MII oocytes. Moreover, the cytoplasmic polyadenylation of dormant Btg4 and Cnot7 mRNAs, which encode key components of deadenylation machinery, is impaired in in vitro MII oocytes, contributing to reduced translation of these deadenylase machinery components and subsequently impaired global maternal mRNA deadenylation. Our findings highlight impaired maternal mRNA deadenylation as a distinct molecular defect in in vitro MII oocytes.
Assuntos
Oócitos , Poliadenilação , Oócitos/metabolismo , Animais , Humanos , Feminino , Camundongos , Poli A/metabolismo , Técnicas de Maturação in Vitro de Oócitos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Transcriptoma , RNA Mensageiro Estocado/metabolismo , RNA Mensageiro Estocado/genética , Metáfase , Exorribonucleases , Proteínas Repressoras , Proteínas de Ciclo CelularRESUMO
La-related proteins (LARPs) are a family of RNA-binding proteins that share a conserved La motif (LaM) domain. LARP1 plays a role in regulating ribosomal protein synthesis and stabilizing mRNAs and has a unique structure without an RNA binding RRM domain adjoining the LaM domain. In this study, we investigated the physical basis for LARP1 specificity for poly(A) sequences and observed an unexpected bias for sequences with single guanines. Multiple guanine substitutions did not increase the affinity, demonstrating preferential recognition of singly guanylated sequences. We also observed that the cyclic di-nucleotides in the cCAS/STING pathway, cyclic-di-GMP and 3',3'-cGAMP, bound with sub-micromolar affinity. Isothermal titration measurements were complemented by high-resolution crystal structures of the LARP1 LaM with six different RNA ligands, including two stereoisomers of a phosphorothioate linkage. The selectivity for singly substituted poly(A) sequences suggests LARP1 may play a role in the stabilizing effect of poly(A) tail guanylation. [Figure: see text].
Assuntos
Poli A , Ligação Proteica , Ribonucleoproteínas , Antígeno SS-B , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Poli A/metabolismo , Poli A/química , Humanos , Modelos Moleculares , Sítios de Ligação , Autoantígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Cristalografia por Raios X , Domínios Proteicos , GMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/química , RNA Mensageiro/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.
Assuntos
Regiões 3' não Traduzidas , Estágios do Ciclo de Vida , Proteínas de Protozoários , Proteínas de Ligação a RNA , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Estágios do Ciclo de Vida/genética , Regiões 3' não Traduzidas/genética , Animais , Transcriptoma , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Poli A/metabolismo , Poli A/genética , PoliadenilaçãoRESUMO
Deamination of bases is a form of DNA damage that occurs spontaneously via the hydrolysis and nitrosation of living cells, generating hypoxanthine from adenine. E. coli endonuclease V (eEndoV) cleaves hypoxanthine-containing double-stranded DNA, whereas human endonuclease V (hEndoV) cleaves hypoxanthine-containing RNA; however, hEndoV in vivo function remains unclear. To date, hEndoV has only been examined using hypoxanthine, because it binds closely to the base located at the cleavage site. Here, we examined whether hEndoV cleaves other lesions (e.g., AP site, 6-methyladenine, xanthine) to reveal its function and whether 2'-nucleoside modification affects its cleavage activity. We observed that hEndoV is hypoxanthine-specific; its activity was the highest with 2'-OH modification in ribose. The cleavage activity of hEndoV was compared based on its base sequence. We observed that it has specificity for adenine located on the 3'-end of hypoxanthine at the cleavage site, both before and after cleavage. These data suggest that hEndoV recognizes and cleaves the inosine generated on the poly A tail to maintain RNA quality. Our results provide mechanistic insight into the role of hEndoV in vivo.
Assuntos
Inosina , Inosina/metabolismo , Humanos , Poli A/metabolismo , Especificidade por Substrato , Hipoxantina/metabolismo , Hipoxantina/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/químicaRESUMO
3'-end cleavage and polyadenylation is an essential process for eukaryotic mRNA maturation. In yeast species, the polyadenylation signals that recruit the processing machinery are degenerate and remain poorly characterized compared with the well-defined regulatory elements in mammals. Here we address this issue by developing deep learning models to deconvolute degenerate cis-regulatory elements and quantify their positional importance in mediating yeast poly(A) site formation, cleavage heterogeneity, and strength. In S. cerevisiae, cleavage heterogeneity is promoted by the depletion of U-rich elements around poly(A) sites as well as multiple occurrences of upstream UA-rich elements. Sites with high cleavage heterogeneity show overall lower strength. The site strength and tandem site distances modulate alternative polyadenylation (APA) under the diauxic stress. Finally, we develop a deep learning model to reveal the distinct motif configuration of S. pombe poly(A) sites, which show more precise cleavage than S. cerevisiae Altogether, our deep learning models provide unprecedented insights into poly(A) site formation of yeast species, and our results highlight divergent poly(A) signals across distantly related species.
Assuntos
Aprendizado Profundo , Poliadenilação , Saccharomyces cerevisiae , Schizosaccharomyces , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Poli A/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
Polyadenylation controls mRNA biogenesis, nucleo-cytoplasmic export, translation and decay. These processes are interdependent and coordinately regulated by poly(A)-binding proteins (PABPs), yet how PABPs are themselves regulated is not fully understood. Here, we report the discovery that human nuclear PABPN1 is phosphorylated by mitotic kinases at four specific sites during mitosis, a time when nucleoplasm and cytoplasm mix. To understand the functional consequences of phosphorylation, we generated a panel of stable cell lines inducibly over-expressing PABPN1 with point mutations at these sites. Phospho-inhibitory mutations decreased cell proliferation, highlighting the importance of PABPN1 phosphorylation in cycling cells. Dynamic regulation of poly(A) tail length and RNA stability have emerged as important modes of gene regulation. We therefore employed long-read sequencing to determine how PABPN1 phospho-site mutants affected poly(A) tails lengths and TimeLapse-seq to monitor mRNA synthesis and decay. Widespread poly(A) tail lengthening was observed for phospho-inhibitory PABPN1 mutants. In contrast, expression of phospho-mimetic PABPN1 resulted in shorter poly(A) tails with increased non-A nucleotides, in addition to increased transcription and reduced stability of a distinct cohort of mRNAs. Taken together, PABPN1 phosphorylation remodels poly(A) tails and increases mRNA turnover, supporting the model that enhanced transcriptome dynamics reset gene expression programs across the cell cycle.
Assuntos
Mitose , Poli A , Proteína I de Ligação a Poli(A) , Poliadenilação , Estabilidade de RNA , RNA Mensageiro , Transcriptoma , Humanos , Mitose/genética , Proteína I de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Fosforilação , Poli A/metabolismo , Estabilidade de RNA/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Células HeLaRESUMO
Stress induces global stabilization of the mRNA poly(A) tail (PAT) and the assembly of untranslated poly(A)-tailed mRNA into mRNPs that accumulate in stress granules (SGs). While the mechanism behind stress-induced global PAT stabilization has recently emerged, the biological significance of PAT stabilization under stress remains elusive. Here, we demonstrate that stress-induced PAT stabilization is a prerequisite for SG formation. Perturbations in PAT length impact SG formation; PAT shortening, achieved by overexpressing mRNA deadenylases, inhibits SG formation, whereas PAT lengthening, achieved by overexpressing their dominant negative mutants or downregulating deadenylases, promotes it. PABPC1, which specifically binds to the PAT, is crucial for SG formation. Complementation analyses reveal that the PABC/MLLE domain of PABPC1, responsible for binding PAM2 motif-containing proteins, plays a key role. Among them, ataxin-2 is a known SG component. A dominant-negative approach reveals that the PAM2 motif of ataxin-2 is essential for SG formation. Notably, ataxin-2 increases stress sensitivity, lowering the threshold for SG formation, probably by promoting the aggregation of PABPC1-bound mRNA. The C-terminal region is responsible for the self-aggregation of ataxin-2. These findings underscore the critical roles of mRNA PAT, PABPC1 and ataxin-2 in SG formation and provide mechanistic insights into this process.
Assuntos
Ataxina-2 , Poli A , Proteína I de Ligação a Poli(A) , RNA Mensageiro , Grânulos de Estresse , Proteína I de Ligação a Poli(A)/metabolismo , Proteína I de Ligação a Poli(A)/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Ataxina-2/metabolismo , Ataxina-2/genética , Humanos , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genética , Poli A/metabolismo , Ligação Proteica , Estabilidade de RNA , Células HeLa , Estresse Fisiológico/genéticaRESUMO
Poly(A) tails are crucial for mRNA translation and degradation, but the exact relationship between tail length and mRNA kinetics remains unclear. Here, we employ a small library of identical mRNAs that differ only in their poly(A)-tail length to examine their behavior in human embryonic kidney cells. We find that tail length strongly correlates with mRNA degradation rates but is decoupled from translation. Interestingly, an optimal tail length of â¼100 nt displays the highest translation rate, which is identical to the average endogenous tail length measured by nanopore sequencing. Furthermore, poly(A)-tail length variability-a feature of endogenous mRNAs-impacts translation efficiency but not mRNA degradation rates. Stochastic modeling combined with single-cell tracking reveals that poly(A) tails provide cells with an independent handle to tune gene expression fluctuations by decoupling mRNA degradation and translation. Together, this work contributes to the basic understanding of gene expression regulation and has potential applications in nucleic acid therapeutics.
Assuntos
Poli A , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Poli A/metabolismo , Poli A/genética , Biossíntese de Proteínas/genética , Estabilidade de RNA/genética , Células HEK293 , Regulação da Expressão Gênica/genéticaRESUMO
Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.
Assuntos
Poli A , Poliadenilação , Análise de Célula Única , Análise de Célula Única/métodos , Humanos , Poli A/metabolismo , Animais , Camundongos , Íntrons/genética , Transcriptoma/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Regulação da Expressão GênicaRESUMO
The poly(A) tail plays an important role in maintaining mRNA stability and influences translation efficiency via binding with PABP. However, the impact of poly(A) tail length on mRNA translation remains incompletely understood. This study explores the effects of poly(A) tail length on human translation. We determined the translation rates in cell lysates using mRNAs with different poly(A) tails. Cap-dependent translation was stimulated by the poly(A) tail, however, it was largely independent of poly(A) tail length, with an exception observed in the case of the 75 nt poly(A) tail. Conversely, cap-independent translation displayed a positive correlation with poly(A) tail length. Examination of translation stages uncovered the dependence of initiation and termination on the presence of the poly(A) tail, but the efficiency of initiation remained unaffected by poly(A) tail extension. Further study unveiled that increased binding of eRFs to the ribosome with the poly(A) tail extension induced more efficient hydrolysis of peptidyl-tRNA. Building upon these findings, we propose a crucial role for the 75 nt poly(A) tail in orchestrating the formation of a double closed-loop mRNA structure within human cells which couples the initiation and termination phases of translation.
Assuntos
Poli A , Biossíntese de Proteínas , RNA Mensageiro , Ribossomos , Humanos , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Poli A/metabolismo , Ribossomos/metabolismo , Ribossomos/genética , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , Células HeLa , Capuzes de RNA/metabolismo , Estabilidade de RNARESUMO
Microscopic understanding of protein-RNA interactions is important for different biological activities, such as RNA transport, translation, splicing, silencing, etc. Polyadenine (Poly(A)) binding proteins (PABPs) make up a class of regulatory proteins that play critical roles in protecting the poly(A) tails of cellular mRNAs from nuclease degradation. In this work, we performed molecular dynamics simulations to investigate the conformational modifications of human PABP protein and poly(A) RNA that occur during complexation. It is demonstrated that the intermediate linker domain of the protein transforms from a disordered coil-like structure to a helical form during the recognition process, leading to the formation of the complex. On the other hand, disordered collapsed coil-like RNA on complexation has been found to transform into a rigid extended conformation. Importantly, the binding free energy calculation showed that the thermodynamic stability of the complex is primarily guided by favorable hydrophobic interactions between the protein and the RNA.
Assuntos
Simulação de Dinâmica Molecular , Poli A , Proteínas de Ligação a Poli(A) , Termodinâmica , Humanos , Poli A/química , Poli A/metabolismo , Proteínas de Ligação a Poli(A)/química , Proteínas de Ligação a Poli(A)/metabolismo , Conformação Proteica , Ligação Proteica , Interações Hidrofóbicas e Hidrofílicas , RNA/química , RNA/metabolismoRESUMO
The RNA polymerase II (Pol II) elongation rate influences poly(A) site selection, with slow and fast Pol II derivatives causing upstream and downstream shifts, respectively, in poly(A) site utilization. In yeast, depletion of either of the histone chaperones FACT or Spt6 causes an upstream shift of poly(A) site use that strongly resembles the poly(A) profiles of slow Pol II mutant strains. Like slow Pol II mutant strains, FACT- and Spt6-depleted cells exhibit Pol II processivity defects, indicating that both Spt6 and FACT stimulate the Pol II elongation rate. Poly(A) profiles of some genes show atypical downstream shifts; this subset of genes overlaps well for FACT- or Spt6-depleted strains but is different from the atypical genes in Pol II speed mutant strains. In contrast, depletion of histone H3 or H4 causes a downstream shift of poly(A) sites for most genes, indicating that nucleosomes inhibit the Pol II elongation rate in vivo. Thus, chromatin-based control of the Pol II elongation rate is a potential mechanism, distinct from direct effects on the cleavage/polyadenylation machinery, to regulate alternative polyadenylation in response to genetic or environmental changes.
Assuntos
Cromatina , Histonas , Poliadenilação , RNA Polimerase II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Cromatina/metabolismo , Cromatina/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Elongação da Transcrição Genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Poli A/metabolismoRESUMO
Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.
Assuntos
Genoma Viral , Herpesvirus Humano 1 , Proteínas Imediatamente Precoces , Mutação , RNA Polimerase II , Transcrição Viral , Animais , Humanos , Linhagem Celular , Chlorocebus aethiops , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Genes Virais/genética , Genoma Viral/genética , Herpes Simples/virologia , Herpes Simples/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/deficiência , Proteínas Imediatamente Precoces/genética , Poli A/genética , Poli A/metabolismo , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Células Vero , Transcrição Viral/efeitos dos fármacos , Transcrição Viral/genética , Replicação Viral/genéticaRESUMO
RNA plays an important role in many biological processes which are crucial for cell survival, and it has been suggested that it may be possible to inhibit individual processes involved in many diseases by targeting specific sequences of RNA. The aim of this work is to determine the affinity of novel 3,9-disubstited acridine derivative 1 with three different RNA molecules, namely single stranded poly(rA), double stranded homopolymer poly(rAU) and triple stranded poly(rUAU). The results of the absorption titration assays show that the binding constant of the novel derivative to the RNA molecules was in the range of 1.7-6.2 × 104 mol dm-3. The fluorescence and circular dichroism titration assays revealed considerable changes. The most significant results in terms of interpreting the nature of the interactions were the melting temperatures of the RNA samples in complexes with the 1. In the case of poly(rA), denaturation resulted in a self-structure formation; increased stabilization was observed for poly(rAU), while the melting points of the ligand-poly(rUAU) complex showed significant destabilization as a result of the interaction. The principles of molecular mechanics were applied to propose the non-bonded interactions within the binding complex, pentariboadenylic acid and acridine ligand as the study model. Initial molecular docking provided the input structure for advanced simulation techniques. Molecular dynamics simulation and cluster analysis reveal π - π stacking and the hydrogen bonds formation as the main forces that can stabilize the binding complex. Subsequent MM-GBSA calculations showed negative binding enthalpy accompanied the complex formation and proposed the most preferred conformation of the interaction complex.
Assuntos
Acridinas , Dicroísmo Circular , Simulação de Acoplamento Molecular , Poli A , Acridinas/química , Acridinas/metabolismo , Poli A/química , Poli A/metabolismo , Termodinâmica , Espectrometria de Fluorescência , RNA/química , RNA/metabolismo , Conformação de Ácido NucleicoRESUMO
Most eukaryotic mRNAs and different non-coding RNAs undergo a form of 3' end processing known as polyadenylation. Polyadenylation machinery is present in almost all organisms except few species. In bacteria, the machinery has evolved from PNPase, which adds heteropolymeric tails, to a poly(A)-specific polymerase. Differently, a complex machinery for accurate polyadenylation and several non-canonical poly(A) polymerases are developed in eukaryotes. The role of poly(A) tail has also evolved from serving as a degradative signal to a stabilizing modification that also regulates translation. In this review, we discuss poly(A) tail emergence in prokaryotes and its development into a stable, yet dynamic feature at the 3' end of mRNAs in eukaryotes. We also describe how appearance of novel poly(A) polymerases gives cells flexibility to shape poly(A) tail. We explain how poly(A) tail dynamics help regulate cognate RNA metabolism in a context-dependent manner, such as during oocyte maturation. Finally, we describe specific mRNAs in metazoans that bear stem-loops instead of poly(A) tails. We conclude with how recent discoveries about poly(A) tail can be applied to mRNA technology. This article is categorized under: RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Processing > 3' End Processing RNA Turnover and Surveillance > Regulation of RNA Stability.
Assuntos
Poli A , Poliadenilação , Poli A/genética , Poli A/metabolismo , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Eucariotos/genética , Eucariotos/metabolismoRESUMO
We have previously introduced the first generation of C3P3, an artificial system that allows the autonomous in-vivo production of mRNA with m7GpppN-cap. While C3P3-G1 synthesized much larger amounts of capped mRNA in human cells than conventional nuclear expression systems, it produced a proportionately much smaller amount of the corresponding proteins, indicating a clear defect of mRNA translatability. A possible mechanism for this poor translatability could be the rudimentary polyadenylation of the mRNA produced by the C3P3-G1 system. We therefore sought to develop the C3P3-G2 system using an artificial enzyme to post-transcriptionally lengthen the poly(A) tail. This system is based on the mutant mouse poly(A) polymerase alpha fused at its N terminus with an N peptide from the λ virus, which binds to BoxBr sequences placed in the 3'UTR region of the mRNA of interest. The resulting system selectively brings mPAPαm7 to the target mRNA to elongate its poly(A)-tail to a length of few hundred adenosine. Such elongation of the poly(A) tail leads to an increase in protein expression levels of about 2.5-3 times in cultured human cells compared to the C3P3-G1 system. Finally, the coding sequence of the tethered mutant poly(A) polymerase can be efficiently fused to that of the C3P3-G1 enzyme via an F2A sequence, thus constituting the single-ORF C3P3-G2 enzyme. These technical developments constitute an important milestone in improving the performance of the C3P3 system, paving the way for its applications in bioproduction and non-viral human gene therapy.
Assuntos
RNA Polimerases Dirigidas por DNA , Poliadenilação , Animais , Humanos , Camundongos , RNA Polimerases Dirigidas por DNA/genética , RNA Mensageiro/metabolismo , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , Poli A/genética , Poli A/metabolismoRESUMO
During oocyte maturation and early embryogenesis, changes in mRNA poly(A)-tail lengths strongly influence translation, but how these tail-length changes are orchestrated has been unclear. Here, we performed tail-length and translational profiling of mRNA reporter libraries (each with millions of 3' UTR sequence variants) in frog oocytes and embryos and in fish embryos. Contrasting to previously proposed cytoplasmic polyadenylation elements (CPEs), we found that a shorter element, UUUUA, together with the polyadenylation signal (PAS), specify cytoplasmic polyadenylation, and we identified contextual features that modulate the activity of both elements. In maturing oocytes, this tail lengthening occurs against a backdrop of global deadenylation and the action of C-rich elements that specify tail-length-independent translational repression. In embryos, cytoplasmic polyadenylation becomes more permissive, and additional elements specify waves of stage-specific deadenylation. Together, these findings largely explain the complex tapestry of tail-length changes observed in early frog and fish development, with strong evidence of conservation in both mice and humans.