RESUMO
Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3' ends of CRISPR-Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3' ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3' ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.
Assuntos
Edição de Genes , Proteínas de Ligação a RNA , Humanos , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Células K562 , Poli U/genética , Poli U/metabolismo , RNA Polimerase III/metabolismo , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Mobile genetic elements threaten genome integrity in all organisms. RDE-3 (also known as MUT-2) is a ribonucleotidyltransferase that is required for transposon silencing and RNA interference in Caenorhabditis elegans1-4. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3' termini of these RNAs (designated poly(UG) or pUG tails)5. Here we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to targets of RNA interference, as well as to transposon RNAs. RNA fragments attached to pUG tails with more than 16 perfectly alternating 3' U and G nucleotides become gene-silencing agents. pUG tails promote gene silencing by recruiting RNA-dependent RNA polymerases, which use pUG-tailed RNAs (pUG RNAs) as templates to synthesize small interfering RNAs (siRNAs). Our results show that cycles of pUG RNA-templated siRNA synthesis and siRNA-directed pUG RNA biogenesis underlie double-stranded-RNA-directed transgenerational epigenetic inheritance in the C. elegans germline. We speculate that this pUG RNA-siRNA silencing loop enables parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/parasitologia , Epigênese Genética/genética , Genoma/genética , Hereditariedade , Poli G/genética , Poli U/genética , RNA Mensageiro/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Masculino , Nucleotidiltransferases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Polimerase Dependente de RNA/metabolismo , Moldes GenéticosRESUMO
The phase separation of protein and RNA mixtures underpins the assembly and regulation of numerous membraneless organelles in cells. The ubiquity of protein-RNA condensates in cellular regulatory processes is in part due to their sensitivity to RNA concentration, which affects their physical properties and stability. Recent experiments with poly-cationic peptide-RNA mixtures have revealed closed-loop phase diagrams featuring lower and upper critical solution temperatures. These diagrams indicate reentrant phase transitions shaped by biomolecular interactions and entropic forces such as solvent and ion reorganization. We employed atomistic simulations to study mixtures with various RNA-polylysine stoichiometries and temperatures to elucidate the microscopic driving forces behind reentrant phase transitions in protein-RNA mixtures. Our findings reveal an intricate interplay between hydration, ion condensation, and specific RNA-polylysine hydrogen bonding, resulting in distinct stoichiometry-dependent phase equilibria governing stabilities and structures of the condensate phase. Our simulations show that reentrant transitions are accompanied by desolvation around the phosphate groups of RNA, with increased contacts between phosphate and lysine side chains. In RNA-rich systems at lower temperatures, RNA molecules can form an extensive pi-stacking and hydrogen bond network, leading to percolation. In protein-rich systems, no such percolation-induced transitions are observed. Furthermore, we assessed the performance of three prominent water force fields-Optimal Point Charge (OPC), TIP4P-2005, and TIP4P-D-in capturing reentrant phase transitions. OPC provided a superior balance of interactions, enabling effective capture of reentrant transitions and accurate characterization of changes in solvent reorganization. This study offers atomistic insights into the nature of reentrant phase transitions using simple model peptide and nucleotide mixtures. We believe that our results are broadly applicable to larger classes of peptide-RNA mixtures exhibiting reentrant phase transitions.
Assuntos
Simulação de Dinâmica Molecular , Transição de Fase , Polilisina , RNA , Polilisina/química , RNA/química , Ligação de Hidrogênio , Poli U/químicaRESUMO
RNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines, and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we used a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RBPs. Half of the analyzed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster end binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1's RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather nonspecific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Associadas à Distrofina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Tiorredoxinas/metabolismo , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Associadas à Distrofina/química , Proteínas Associadas à Distrofina/genética , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Ovário/citologia , Ovário/metabolismo , Poli U/química , Poli U/genética , Poli U/metabolismo , Ligação Proteica , RNA/química , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiorredoxinas/química , Tiorredoxinas/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/química , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genéticaRESUMO
Two arene ruthenium(II) complexes [η6-(C6H6)Ru(pprip)Cl]PF6 (Ru1; pprip = 2-(3-phenyl-1H-pyrazol-4-yl)-imidazolo[4,5-f][1,10]phenanthroline) and [η6-(C6H6)Ru(H2iiP)Cl]PF6 (Ru2; H2iiP = 2-(indole-3-yl)-imidazolo[4,5-f][1,10]phenanthroline) have been synthesized and characterized in this work. Binding properties of Ru1 and Ru2 with the triplex RNA poly(U)â¢poly(A)*poly(U) were investigated by spectrophotometry and spectrofluorometry as well as viscosimetry. Analysis of spectroscopic titrations and viscosity measurements show that the two complexes bind with the triplex through intercalation, while the binding affinity for Ru2 toward the triplex is stronger than that for Ru1. Melting experiments indicate that the stabilizing effects of Ru1 and Ru2 toward the triplex differ from each other. Under the conditions used herein, Ru1 only stabilizes the Hoogsteen base-paired strand (third strand) without affecting stabilization of the Watson-Crick base-paired strand (the template duplex) of the triplex, while Ru2 stabilizes both the template duplex and the third strand. Although the two complexes prefer to stabilizing the third strand rather than the template duplex, the third-strand stabilization effect of Ru2 is stronger than that of Ru1. The obtained results of this work reveal that the planarity of the intercalative ligands plays an important role in the triplex stabilization by arene Ru(II) complexes.
Assuntos
Poli A , Rutênio , Poli A/química , Rutênio/química , Poli U/química , RNA/química , Fenantrolinas , Conformação de Ácido Nucleico , Espectrometria de FluorescênciaRESUMO
Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)â poly(A)*poly(U) have been investigated by various biophysical techniques. Spectrophotometric titrations and viscosity measurements suggested that enantiomers Λ-1 and Δ-1 bind with the triplex through intercalation, while the binding strengths of the two enantiomers toward the triplex differed only slightly from each other. Fluorescence titrations showed that although enantiomers Λ-1 and Δ-1 exhibited molecular "light switch" effects toward the triplex, the effect of Δ-1 was more marked. Furthermore, Furthermore, thermal denaturation showed that the two enantiomers have significantly different stabilizing effects on the triplex. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar to a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex. Two chiral ruthenium(II) polypyridyl complexes, Λ-[Ru(bpy)2(dppx)]2+ (bpy = 2,2'-bipyridine, dppx = 7,8-dimethyldipyridophenazine; Λ-1) and Δ-[Ru(bpy)2(dppx)]2+ (Δ-1) have been synthesized and characterized in this work. Interactions of Λ-1 and Δ-1 with the RNA triplex poly(U)â poly(A)*poly(U) have been investigated by various biophysical techniques. The obtained results indicate that the racemic complex [Ru(bpy)2(dppx)]2+ is similar as a non-specific metallointercalator for the triplex investigated in this study, and chiralities of Ru(II) polypyridine complexes have an important influence on the binding and stabilizing effects of enantiomers toward the triplex.
Assuntos
Poli A , Rutênio , Poli A/química , Rutênio/química , Poli U/química , 2,2'-Dipiridil , RNA/químicaRESUMO
Coronaviruses (CoVs) are positive-sense RNA viruses that can emerge from endemic reservoirs and infect zoonotically, causing significant morbidity and mortality. CoVs encode an endoribonuclease designated EndoU that facilitates evasion of host pattern recognition receptor MDA5, but the target of EndoU activity was not known. Here, we report that EndoU cleaves the 5'-polyuridines from negative-sense viral RNA, termed PUN RNA, which is the product of polyA-templated RNA synthesis. Using a virus containing an EndoU catalytic-inactive mutation, we detected a higher abundance of PUN RNA in the cytoplasm compared to wild-type-infected cells. Furthermore, we found that transfecting PUN RNA into cells stimulates a robust, MDA5-dependent interferon response, and that removal of the polyuridine extension on the RNA dampens the response. Overall, the results of this study reveal the PUN RNA to be a CoV MDA5-dependent pathogen-associated molecular pattern (PAMP). We also establish a mechanism for EndoU activity to cleave and limit the accumulation of this PAMP. Since EndoU activity is highly conserved in all CoVs, inhibiting this activity may serve as an approach for therapeutic interventions against existing and emerging CoV infections.
Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Coronavirus/metabolismo , Endorribonucleases/metabolismo , Poli U/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Antivirais/farmacologia , Linhagem Celular , Chlorocebus aethiops , Coronavirus/enzimologia , Coronavirus/imunologia , Endorribonucleases/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Interferons/farmacologia , Poli U/química , RNA Viral/genética , RNA Viral/metabolismo , Uridina/química , Células Vero , Proteínas não Estruturais Virais/genética , Replicação Viral/fisiologiaRESUMO
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains five CCCH domains and one CCHC domain and recognizes two conserved 3' pre-mRNA sequences: an AU hexamer and a U-rich motif. AU hexamer motifs are common in pre-mRNAs and are typically defined as AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs and can affect polyadenylation efficiency or be linked to diseases. The effects of disease-related variations on CPSF30/pre-mRNA binding were determined using a construct of CPSF30 that contains just the five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify the variability within the AU hexamer sequence in pre-mRNAs. The effects of this sequence variability on CPSF30-5F/RNA binding affinities were measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer were found to be important for RNA binding. Bioinformatics revealed that the three bases flanking the AU hexamer at the 5' and 3' ends are twice as likely to be adenine or uracil as guanine and cytosine. The presence of A and U residues in these flanking regions was determined to promote higher-affinity CPSF30-5F/RNA binding than G and C residues. The addition of the zinc knuckle domain to CPSF30-5F (CPSF30-FL) restored binding to AU hexamer variants. This restoration of binding is connected to the presence of a U-rich sequence within the pre-mRNA to which the zinc knuckle binds. A mechanism of differential RNA binding by CPSF30, modulated by accessibility of the two RNA binding sites, is proposed.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Poli U/metabolismo , Poliadenilação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Animais , Sítios de Ligação , Bovinos , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Ligação Proteica , Precursores de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Dedos de ZincoRESUMO
Low-virulence classical swine fever virus (CSFV) strains make CSF eradication particularly difficult. Few data are available on the molecular determinants of CSFV virulence. The aim of the present study was to assess a possible role for CSFV virulence of a unique, uninterrupted 36-uridine (poly-U) sequence found in the 3' untranslated region (3' UTR) of the low-virulence CSFV isolate Pinar de Rio (PdR). To this end, a pair of cDNA-derived viruses based on the PdR backbone were generated, one carrying the long poly-U insertion in the 3' UTR (vPdR-36U) and the other harboring the standard 5 uridines at this position (vPdR-5U). Two groups of 20 5-day-old piglets were infected with vPdR-36U and vPdR-5U. Ten contact piglets were added to each group. Disease progression, virus replication, and immune responses were monitored for 5 weeks. The vPdR-5U virus was significantly more virulent than the vPdR-36U virus, with more severe disease, higher mortality, and significantly higher viral loads in serum and body secretions, despite similar replication characteristics in cell culture. The two viruses were transmitted to all contact piglets. Ninety percent of the piglets infected with vPdR-36U seroconverted, while only one vPdR-5U-infected piglet developed antibodies. The vPdR-5U-infected piglets showed only transient alpha interferon (IFN-α) responses in serum after 1 week of infection, while the vPdR-36U-infected piglets showed sustained IFN-α levels during the first 2 weeks. Taken together, these data show that the 3' UTR poly-U insertion acquired by the PdR isolate reduces viral virulence and activates the innate and humoral immune responses without affecting viral transmission.IMPORTANCE Classical swine fever (CSF), a highly contagious viral disease of pigs, is still endemic in some countries of Asia and Central and South America. Considering that the 3' untranslated region (3' UTR) plays an important role in flavivirus replication, the present study showed for the first time that a long polyuridine sequence acquired in the 3' UTR by an endemic CSFV isolate can activate immunity, control viral replication, and modulate disease in piglets. Our findings provide new avenues for the development of novel vaccines against infections with CSF virus and other flaviviruses. Knowledge of molecular virulence determinants is also relevant for future development of rapid and efficient diagnostic tools for the prediction of the virulence of field isolates and for efficient CSF control.
Assuntos
Regiões 3' não Traduzidas/imunologia , Vírus da Febre Suína Clássica , Peste Suína Clássica , Mutagênese Insercional , Poli U , RNA Viral , Animais , Peste Suína Clássica/genética , Peste Suína Clássica/imunologia , Peste Suína Clássica/patologia , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/imunologia , Vírus da Febre Suína Clássica/patogenicidade , Humanos , Interferon-alfa/imunologia , Poli U/genética , Poli U/imunologia , RNA Viral/genética , RNA Viral/imunologia , SuínosRESUMO
BACKGROUND: Peritoneal metastases portend poor prognosis in the setting of standard chemotherapy. Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) improves outcomes, but relapse is common. We report a phase II trial evaluating the safety and efficacy of adjuvant αDC1 vaccination with chemokine modulation (CKM) after CRS/HIPEC. METHODS: Patients undergoing CRS/HIPEC for appendiceal cancer, colorectal cancer, or peritoneal mesothelioma were enrolled. In addition to standard adjuvant chemotherapy, patients received intranodal and intradermal injections of autologous tumor-loaded αDC1 vaccine. After each vaccine booster, patients received CKM over 4 days, consisting of celecoxib, interferon (IFN)-α, and rintatolimod. RESULTS: Forty-six patients underwent CRS/HIPEC followed by αDC1 treatment, including 24 appendiceal primaries, 20 colorectal, and 2 mesotheliomas. DC maturation was successful, with 97% expressing HLA-DR and CD86. Tumor cell recovery from peritoneal tumors was challenging, resulting in only 17% of patients receiving the target dose of αDC1. The αDC1 and CKM regimen was well tolerated. CKM successfully modulated serum inflammatory cytokine and chemokine levels. Median progression-free survival (PFS) for appendiceal primaries was 50.4, 34.2, and 8.9 months for grade 1, 2, and 3 tumors, respectively, while median PFS for colorectal cancer was 20.5 and 8.9 months for moderately and poorly differentiated tumors, respectively. CONCLUSIONS: Adjuvant autologous tumor antigen-loaded αDC1 vaccine and CKM is well tolerated. The mucinous nature of peritoneal metastases limits the feasibility of obtaining adequate autologous tumor cells. The improvement in median PFS did not meet our predefined thresholds, leading us to conclude that αDC1 vaccination is not appropriate for patients undergoing CRS/HIPEC for peritoneal metastases.
Assuntos
Neoplasias Colorretais , Hipertermia Induzida , Neoplasias Peritoneais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Celecoxib/uso terapêutico , Neoplasias Colorretais/terapia , Procedimentos Cirúrgicos de Citorredução , Células Dendríticas , Humanos , Quimioterapia Intraperitoneal Hipertérmica , Interferon-alfa/uso terapêutico , Recidiva Local de Neoplasia , Neoplasias Peritoneais/tratamento farmacológico , Poli I-C , Poli URESUMO
TUT4 and the closely related TUT7 are non-templated poly(U) polymerases required at different stages of development, and their mis-regulation or mutation has been linked to important cancer pathologies. While TUT4(7) interaction with its pre-miRNA targets has been characterized in detail, the molecular bases of the broader target recognition process are unclear. Here, we examine RNA binding by the ZnF domains of the protein. We show that TUT4(7) ZnF2 contains two distinct RNA binding surfaces that are used in the interaction with different RNA nucleobases in different targets, i.e that this small domain encodes diversity in TUT4(7) selectivity and molecular function. Interestingly and unlike other well-characterized CCHC ZnFs, ZnF2 is not physically coupled to the flanking ZnF3 and acts independently in miRNA recognition, while the remaining CCHC ZnF of TUT4(7), ZnF1, has lost its intrinsic RNA binding capability. Together, our data suggest that the ZnFs of TUT4(7) are independent units for RNA and, possibly, protein-protein interactions that underlay the protein's functional flexibility and are likely to play an important role in building its interaction network.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Epistasia Genética , Regulação da Expressão Gênica , MicroRNAs/genética , Proteínas de Ligação a RNA/metabolismo , Dedos de Zinco , Composição de Bases , Proteínas de Ligação a DNA/química , Humanos , Espectroscopia de Ressonância Magnética , MicroRNAs/química , MicroRNAs/metabolismo , Poli U , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Relação Estrutura-AtividadeRESUMO
Hybrids formed by DNA/RNA and graphene family nanomaterials are considered as potentially useful multifunctional agents in biosensing and nanomedicine. In this work, we study the noncovalent interaction between double-stranded (ds) RNA, polyadenylic:polyuridylic acids (poly(A:U)) and graphene oxide/graphene (GO/Gr) using UV absorption spectroscopy and molecular dynamics (MD) simulations. RNA melting showed that relatively long ds-RNA is adsorbed onto GO (at an ionic strength of [Formula: see text]) at that a large fraction of RNA maintains the duplex structure. It was revealed that this fraction decreases over long time (during a few days), indicating a slow adsorption process of the long polymer. MD simulations showed that the adsorption of duplex (rA)[Formula: see text]: (rU)[Formula: see text] or (rA)[Formula: see text]: (rU)[Formula: see text] on graphene starts with the interaction between [Formula: see text]-systems of graphene and base pairs located at a duplex tail. In contrast to relatively long duplex (rA)[Formula: see text]: (rU)[Formula: see text] which keeps parallel arrangement along the graphene surface, the shorter one ((rA)[Formula: see text]: (rU)[Formula: see text]) always adopts a perpendicular orientation relative to graphene even in case of the initial parallel orientation. It was found out that (rA)[Formula: see text]: (rU)[Formula: see text] forms the stable hybrid with graphene keeping essential fraction of the duplex, while (rA)[Formula: see text]: (rU)[Formula: see text] demonstrates the duplex unzipping into two single strands with time. The interaction energies between adenine/uracil stacked with graphene as well between nucleotides in water environment were determined.
Assuntos
Grafite/química , Nanoestruturas/química , Poli A/química , Polinucleotídeos/química , RNA de Cadeia Dupla/química , Adsorção , Simulação de Dinâmica Molecular , Concentração Osmolar , Poli U/química , Propriedades de SuperfícieRESUMO
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a "zinc finger" protein that plays a crucial role in the transition of pre-mRNA to RNA. CPSF30 contains five conserved CCCH domains and a CCHC "zinc knuckle" domain. CPSF30 activity is critical for pre-mRNA processing. A truncated form of the protein, in which only the CCCH domains are present, has been shown to specifically bind AU-rich pre-mRNA targets; however, the RNA binding and recognition properties of full-length CPSF30 are not known. Herein, we report the isolation and biochemical characterization of full-length CPSF30. We report that CPSF30 contains one 2Fe-2S cluster in addition to five zinc ions, as measured by inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, and X-ray absorption spectroscopy. Utilizing fluorescence anisotropy RNA binding assays, we show that full-length CPSF30 has high binding affinity for two types of pre-mRNA targets, AAUAAA and polyU, both of which are conserved sequence motifs present in the majority of pre-mRNAs. Binding to the AAUAAA motif requires that the five CCCH domains of CPSF30 be present, whereas binding to polyU sequences requires the entire, full-length CPSF30. These findings implicate the CCHC "zinc knuckle" present in the full-length protein as being critical for mediating polyU binding. We also report that truncated forms of the protein, containing either just two CCCH domains (ZF2 and ZF3) or the CCHC "zinc knuckle" domain, do not exhibit any RNA binding, indicating that CPSF30/RNA binding requires several ZF (and/or Fe-S cluster) domains working in concert to mediate RNA recognition.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Poli U/metabolismo , Precursores de RNA/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Cobalto/química , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/genética , Mutação , Ligação Proteica , Precursores de RNA/genética , Zinco/química , Dedos de Zinco , alfa-Sinucleína/genéticaRESUMO
During protein synthesis, the nascent peptide chain traverses the peptide exit tunnel of the ribosome. We monitor the co-translational movement of the nascent peptide using a fluorescent probe attached to the N-terminus of the nascent chain. Due to fluorophore quenching, the time-dependent fluorescence signal emitted by an individual peptide is determined by co-translational events, such as secondary structure formation and peptide-tunnel interactions. To obtain information on these individual events, the measured ensemble fluorescence signal has to be decomposed into position-dependent intensities. Here, we describe mRNA translation as a Markov process with specific fluorescence intensities assigned to the different states of the process. Combining the computed stochastic time evolution of the translation process with a sequence of observed ensemble fluorescence time courses, we compute the unknown position-specific intensities and obtain detailed information on the kinetics of the translation process. In particular, we find that translation of poly(U) mRNAs dramatically slows down at the fourth UUU codon. The method presented here detects subtle differences in the position-specific fluorescence intensities and thus provides a novel approach to study translation kinetics in ensemble experiments.
Assuntos
Códon/genética , Fluorescência , Peptídeos/metabolismo , Biossíntese de Proteínas , Cinética , Cadeias de Markov , Peptídeos/genética , Poli U/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
Understanding of the conformational ensemble of flexible polyelectrolytes, such as single-stranded nucleic acids (ssNAs), is complicated by the interplay of chain backbone entropy and salt-dependent electrostatic repulsions. Molecular elasticity measurements are sensitive probes of the statistical conformation of polymers and have elucidated ssNA conformation at low force, where electrostatic repulsion leads to a strong excluded volume effect, and at high force, where details of the backbone structure become important. Here, we report measurements of ssDNA and ssRNA elasticity in the intermediate-force regime, corresponding to 5- to 100-pN forces and 50-85% extension. These data are explained by a modified wormlike chain model incorporating an internal electrostatic tension. Fits to the elastic data show that the internal tension decreases with salt, from [Formula: see text]5 pN under 5 mM ionic strength to near zero at 1 M. This decrease is quantitatively described by an analytical model of electrostatic screening that ascribes to the polymer an effective charge density that is independent of force and salt. Our results thus connect microscopic chain physics to elasticity and structure at intermediate scales and provide a framework for understanding flexible polyelectrolyte elasticity across a broad range of relative extensions.
Assuntos
DNA de Cadeia Simples/química , Modelos Químicos , Poli U/química , Eletricidade EstáticaRESUMO
We synthesized a new amino acid-fluorescent nucleobase derivative (qAN1-AA) and from it two new fluorescent nucleobase-fluorophore (pyrene) conjugates, whereby only the analogue with the longer and more flexible linker (qAN1-pyr2) self-folded into intramolecularly stacked qAN1/pyrene conformation, yielding characteristic, 100 nm-red-shifted emission (λmax = 500 nm). On the contrary, the shorter and more rigid linker resulted in non-stacked conformation (qAN1-pyr1), characterized by the emission of free pyrene at λmax = 400 nm. Both fluorescent nucleobase-fluorophore (pyrene) conjugates strongly interacted with ds-DNA/RNA grooves with similar affinity but opposite fluorescence response (due to pre-organization), whereas the amino acid-fluorescent base derivative (qAN1-AA) was inactive. However, only intramolecularly self-folded qAN1-pyr2 showed strong fluorescence selectivity toward poly U (Watson-Crick complementary to qAN1 nucleobase) and poly A (reverse Hoogsteen complementary to qAN1 nucleobase), while an opposite emission change was observed for non-complementary poly G and poly C. Non-folded analogue (qAN1-pyr1) showed no ss-RNA selectivity, demonstrating the importance of nucleobase-fluorophore pre-organization.
Assuntos
DNA/química , Corantes Fluorescentes/química , Pirenos/química , Pirenos/síntese química , RNA/química , Dicroísmo Circular , Fluorescência , Ligação de Hidrogênio , Conformação de Ácido Nucleico , Poli A/química , Poli C/química , Poli G/química , Poli U/química , RNA de Cadeia Dupla/química , Solventes/química , Espectrometria de Fluorescência , Água/químicaRESUMO
Unlike double-stranded DNA, single-stranded RNA can be spontaneously packaged into spherical capsids by viral capsid protein (CP) because it is a more compact and flexible polymer. Many systematic investigations of this self-assembly process have been carried out using CP from cowpea chlorotic mottle virus, with a wide range of sequences and lengths of single-stranded RNA. Among these studies are measurements of the relative packaging efficiencies of these RNAs into spherical capsids. In this work, we address a fundamental issue that has received very little attention, namely the question of the preferred curvature of the capsid formed around different RNA molecules. We show in particular that homopolymers of RNA-polyribouridylic acid and polyriboadenylic acid-form exclusively T = 2-sized (â¼22-nm diameter) virus-like particles (VLPs) when mixed with cowpea chlorotic mottle virus CP, independent of their length, ranging from 500 to more than 4000 nucleotides. This is in contrast to "normal-composition" RNAs (i.e., molecules with comparable numbers of each of the four nucleotides and hence capable of developing a large amount of secondary structure because of intramolecular complementarity/basepairing); a curvature corresponding to T = 3-size (â¼28 nm in diameter) is preferred for the VLPs formed with such RNAs. Our work is consistent with the preferred curvature of VLPs being a consequence of interaction of CP with RNA-in particular, the presence or absence of short RNA duplexes-and suggests that the equilibrium size of the capsid results from a trade-off between this optimum size and the cost of confinement.
Assuntos
Bromovirus/química , RNA/química , Concentração de Íons de Hidrogênio , Poli A/química , Poli A/metabolismo , Poli U/química , Poli U/metabolismo , Polimerização , RNA/metabolismoRESUMO
HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.
Assuntos
Primers do DNA/biossíntese , Transcriptase Reversa do HIV/metabolismo , Transcriptase Reversa do HIV/fisiologia , Sequência de Bases , Cristalografia por Raios X/métodos , Primers do DNA/química , DNA Viral , HIV-1/genética , Conformação de Ácido Nucleico , Ácidos Nucleicos , Poli A , Poli U , Polinucleotídeos , Purinas/química , RNA Viral/química , Ribonuclease H/metabolismoRESUMO
Translational control of gene expression plays essential roles in cellular stress responses and organismal development by enabling rapid, selective, and localized control of protein production. Translational regulation depends on context-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. Here, we comprehensively determined the RNA-binding preferences of the eukaryotic initiation factor 4G (eIF4G) to assess whether this core translation initiation factor has intrinsic sequence preferences that may contribute to preferential translation of specific mRNAs. We identified a simple RNA sequence motif-oligo-uridine-that mediates high-affinity binding to eIF4G in vitro. Oligo(U) motifs occur naturally in the transcript leader (TL) of hundreds of yeast genes, and mRNAs with unstructured oligo(U) motifs were enriched in immunoprecipitations against eIF4G. Ribosome profiling following depletion of eIF4G in vivo showed preferentially reduced translation of mRNAs with long TLs, including those that contain oligo(U). Finally, TL oligo(U) elements are enriched in genes with regulatory roles and are conserved between yeast species, consistent with an important cellular function. Taken together, our results demonstrate RNA sequence preferences for a general initiation factor, which cells potentially exploit for translational control of specific mRNAs.
Assuntos
Sítios de Ligação , Fator de Iniciação Eucariótico 4G/metabolismo , Regulação Fúngica da Expressão Gênica , Motivos de Nucleotídeos , Poli U/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequência Conservada , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Rho-independent transcription terminators of the genes encoding bacterial Hfq-binding sRNAs possess a set of seven or more T residues at the 3' end, as noted in previous studies. Here, we have studied the role of the terminator hairpin in the biogenesis of sRNAs focusing on SgrS and RyhB in Escherichia coli. We constructed variant sRNA genes in which the GC-rich inverted repeat sequences are extended to stabilize the terminator hairpins. We demonstrate that the extension of the hairpin stem leads to generation of heterogeneous transcripts in which the poly(U) tail is shortened. The transcripts with shortened poly(U) tails no longer bind to Hfq and lose the ability to repress the target mRNAs. The shortened transcripts are generated in an in vitro transcription system with purified RNA polymerase, indicating that the generation of shortened transcripts is caused by premature transcription termination. We conclude that the terminator structure of sRNA genes is optimized to generate functional sRNAs. Thus, the Rho-independent terminators of sRNA genes possess two common features: a long T residue stretch that is a prerequisite for generation of functional sRNAs and a moderate strength of hairpin structure that ensures the termination at the seventh or longer position within the consecutive T stretch. The modulation of the termination position at the Rho-independent terminators is critical for biosynthesis of functional sRNAs.