RESUMEN
Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , ARN , Ribonucleoproteínas , Edición Génica , Filogenia , Ribonucleoproteínas/genética , ARN Guía de Sistemas CRISPR-Cas/genética , Pliegue de ProteínaRESUMEN
Overgrowth of short tandem repeat sequences in our genes can cause various neurodegenerative disorders. Such repeat sequences are ideal targets for the label-free electrochemical detection of such potential expansions. However, their length- and sequence-dependent secondary structures may interfere with the interfacial charge transfer of a detection platform, making them complex targets. In addition, the gene contains sporadic repeats that may result in false-positive signals. Therefore, it is necessary to design a platform capable of mitigating these effects and ultimately enhancing the specificity of tandem repeats. Here, we analyzed three different backbones of nucleic acid microprobes [DNA, peptide nucleic acid, and lock-nucleic acid (LNA)] to detect in vitro transcribed RNA carrying CAG repeats, which are associated with Huntington's disease, based on the charge-transfer resistance of the interface. We found that the LNA microprobe can distinguish lengths down to the attomolar concentration level and alleviate the effect of secondary structures and sporadic repeats in the sequence, thus distinguishing the "tandem repeats" specifically. Additionally, the control experiments conducted with and without Mg2+ demonstrated the LNA microprobe to perform better in the presence of the divalent cation. The results suggest that the LNA-based platform may eventually lead to the development of a reliable and straightforward biosensor for genetic neurodegenerative disorders.
Asunto(s)
Enfermedad de Huntington , Ácidos Nucleicos , Ácidos Nucleicos de Péptidos , Humanos , Repeticiones de Microsatélite , Enfermedad de Huntington/diagnóstico , Enfermedad de Huntington/genética , Estructura Secundaria de ProteínaRESUMEN
Insufficient tracking of virus introduction, spread, and new lineage emergence for the human monkeypox (mpox) virus 1 (hMPXV1) outbreak of 2022 hindered epidemiological studies and public health response. hMPXV1 mutations accumulated unexpectedly faster than predicted. Thus, new variants with altered pathogenicity could emerge and spread without early detection. Whole genome sequencing addresses this gap when implemented but requires widely accessible and standardized methodologies to be effective both regionally and globally. Here we developed a rapid nanopore whole genome sequencing method complete with working protocols, from DNA extraction to phylogenetic analysis tools. Using this method, we sequenced 84 complete hMPXV1 genomes from Illinois, a Midwestern region of the United States, spanning the first few months of the outbreak. The resulting five-fold increase in hMPXV1 genomes from this region established two previously unnamed global lineages, several mutational profiles not seen elsewhere, multiple separate introductions of the virus into the region, and the likely emergence and spread of new lineages from within this region. These results demonstrate that a dearth of genomic sequencing of hMPXV1 slowed our understanding and response to the mpox outbreak. This accessible nanopore sequencing approach makes near real-time mpox tracking and rapid lineage discovery straightforward and creates a blueprint for how to deploy nanopore sequencing for genomic surveillance of diverse viruses and future outbreaks.
Asunto(s)
Mpox , Secuenciación de Nanoporos , Humanos , Filogenia , Secuenciación Completa del Genoma/métodos , Brotes de EnfermedadesRESUMEN
Methods for rapid and high-throughput screening of transcription in vitro to examine reaction conditions, enzyme mutants, promoter variants, and small molecule modulators can be extremely valuable tools. However, these techniques may be difficult to establish or inaccessible to many researchers. To develop a straightforward and cost-effective platform for assessing transcription in vitro, we used the "Broccoli" RNA aptamer as a direct, real-time fluorescent transcript readout. To demonstrate the utility of our approach, we screened the effect of common reaction conditions and components on bacteriophage T7 RNA polymerase (RNAP) activity using a common quantitative PCR instrument for fluorescence detection. Several essential conditions for in vitro transcription by T7 RNAP were confirmed with this assay, including the importance of enzyme and substrate concentrations, covariation of magnesium and nucleoside triphosphates, and the effects of several typical additives. When we used this method to assess all possible point mutants of a canonical T7 RNAP promoter, our results coincided well with previous reports. This approach should translate well to a broad variety of bacteriophage in vitro transcription systems and provides a platform for developing fluorescence-based readouts of more complex transcription systems in vitro.
Asunto(s)
Aptámeros de Nucleótidos/genética , Bioensayo , ARN Polimerasas Dirigidas por ADN/genética , ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas Virales/genética , Secuencia de Aminoácidos , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Secuencia de Bases , ADN/química , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Magnesio/química , Magnesio/farmacología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación Puntual , Regiones Promotoras Genéticas , Nucleósidos de Purina/química , Nucleósidos de Purina/farmacología , Nucleósidos de Pirimidina/química , Nucleósidos de Pirimidina/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Cloruro de Sodio/química , Cloruro de Sodio/farmacología , Espectrometría de Fluorescencia , Espermidina/química , Espermidina/farmacología , Fracciones Subcelulares/metabolismo , Transcripción Genética , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
We report on the synthesis of siRNAs containing both 2'-5'- and 3'-5'-internucleotide linkages and their effects on siRNA structure, function, and interaction with RNAi proteins. Screening of these siRNAs against their corresponding mRNA targets showed that 2'-5' linkages were well tolerated in the sense strand, but only at a few positions in the antisense strand. Extensive modification of the antisense strand minimally affected 5'-phosphorylation of the siRNA by kinases, however, it negatively affected siRNA loading into human AGO2. Modelling and molecular dynamics simulations were fully consistent with these findings. Furthermore, our studies indicated that the presence of a single 5'p-rN1-(2'-5')-N2 unit in the antisense strand does not alter the 'clover leaf' bend and sugar puckers that are critical for anchoring the 5'-phosphate to Ago 2 MID domain. Importantly, 2'-5'-linkages had the added benefit of abrogating immune-stimulatory activity of siRNAs. Together, these results demonstrate that 2'-5'/3'-5'-modified siRNAs, when properly designed, can offer an efficient new class of siRNAs with diminished immune-stimulatory responses.
Asunto(s)
Interferencia de ARN , ARN Interferente Pequeño/química , Proteínas Argonautas/metabolismo , Conformación de Carbohidratos , Células HeLa , Humanos , Luciferasas de Luciérnaga/genética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/inmunología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
CRISPR (clustered regularly interspaced short palindromic repeat) endonucleases are at the forefront of biotechnology, synthetic biology and gene editing. Methods for controlling enzyme properties promise to improve existing applications and enable new technologies. CRISPR enzymes rely on RNA cofactors to guide catalysis. Therefore, chemical modification of the guide RNA can be used to characterize structure-activity relationships within CRISPR ribonucleoprotein (RNP) enzymes and identify compatible chemistries for controlling activity. Here, we introduce chemical modifications to the sugar-phosphate backbone of Streptococcus pyogenes Cas9 CRISPR RNA (crRNA) to probe chemical and structural requirements. Ribose sugars that promoted or accommodated A-form helical architecture in and around the crRNA 'seed' region were tolerated best. A wider range of modifications were acceptable outside of the seed, especially D-2'-deoxyribose, and we exploited this property to facilitate exploration of greater chemical diversity within the seed. 2'-fluoro was the most compatible modification whereas bulkier O-methyl sugar modifications were less tolerated. Activity trends could be rationalized for selected crRNAs using RNP stability and DNA target binding experiments. Cas9 activity in vitro tolerated most chemical modifications at predicted 2'-hydroxyl contact positions, whereas editing activity in cells was much less tolerant. The biochemical principles of chemical modification identified here will guide CRISPR-Cas9 engineering and enable new or improved applications.
Asunto(s)
Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , ARN Bacteriano/química , División del ADN , ADN de Forma A/química , ARN Bacteriano/metabolismo , Ribonucleoproteínas/metabolismo , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/genética , Relación Estructura-ActividadRESUMEN
Approximately 3% of the human genome is composed of short tandem repeat (STR) DNA sequence known as microsatellites, which can be found in both coding and non-coding regions. When associated with genic regions, expansion of microsatellite repeats beyond a critical threshold causes dozens of neurological repeat expansion disorders. To better understand the molecular pathology of repeat expansion disorders, precise cloning of microsatellite repeat sequence and expansion size is highly valuable. Unfortunately, cloning repeat expansions is often challenging and presents a significant bottleneck to practical investigation. Here, we describe a clear method for seamless and systematic cloning of practically any microsatellite repeat expansion. We use cloning and expansion of GGGGCC repeats, which are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as an example. We employ a recursive directional ligation (RDL) technique to build multiple GGGGCC repeat-containing vectors. We describe methods to validate repeat expansion cloning, including diagnostic restriction digestion, PCR across the repeat, and next-generation long-read MinION nanopore sequencing. Validated cloning of microsatellite repeats beyond the critical expansion threshold can facilitate step-by-step characterization of disease mechanisms at the cellular and molecular level.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Clonación Molecular/métodos , Expansión de las Repeticiones de ADN , Demencia Frontotemporal/genética , Repeticiones de Microsatélite , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Secuencia de Bases , Proteína C9orf72/metabolismo , Enzimas de Restricción del ADN/química , Escherichia coli/genética , Escherichia coli/metabolismo , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Genoma Humano , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Infrared spectroscopy detects the formation of G-quadruplexes in guanine-rich nucleic acid sequences through shifts in the guanine C=O stretch mode. Here, we use ultrafast 2D infrared (IR) spectroscopy and isotope substitution to show that these shifts arise from vibrational delocalization among stacked G-quartets. This provides a direct measure of the sizes of locally ordered motifs in heterogeneous samples with substantial disordered regions. We find that parallel-stranded, potassium-bound DNA G-quadruplexes are limited to five consecutive G-quartets and 3-4 consecutive layers are preferred for longer polyguanine tracts. The resulting potassium-dependent G-quadruplex assembly landscape reflects the polyguanine tract lengths found in genomes, the ionic conditions prevalent in healthy mammalian cells, and the onset of structural disorder in disease states. Our study describes spectral markers that can be used to probe other G-quadruplex structures and provides insight into the fundamental limits of their formation in biological and artificial systems.
Asunto(s)
ADN/química , ADN/síntesis química , G-Cuádruplex , Humanos , Conformación de Ácido Nucleico , Tamaño de la Partícula , Espectrofotometría InfrarrojaRESUMEN
DNA repeat expansion sequences cause a myriad of neurological diseases when they expand beyond a critical threshold. Previous electrochemical approaches focused on the detection of trinucleotide repeats (CAG, CGG, and GAA) and relied on labeling of the probe and/or target strands or enzyme-linked assays. However, detection of expanded GC-rich sequences is challenging because they are prone to forming secondary structures such as cruciforms and quadruplexes. Here, we present label-free detection of hexanucleotide GGGGCC repeat sequences, which cause the leading genetic form of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The approach relies on capturing targets by surface-bound oligonucleotide probes with a different number of complementary repeats, which proportionately translates the length of the target strands into charge transfer resistance (RCT) signal measured by electrochemical impedance spectroscopy. The probe carrying three tandem repeats transduces the number of repeats into RCT with a 3× higher calibration sensitivity and detection limit. Chronocoulometric measurements show a decrease in surface density with increasing repeat length, which is opposite of the impedance trend. This implies that the length of the target itself can contribute to amplification of the impedance signal independent of the surface density. Moreover, the probe can distinguish between a control and patient sequences while remaining insensitive to non-specific Huntington's disease (CAG) repeats in the presence of a complementary target. This label-free strategy might be applied to detect the length of other neurodegenerative repeat sequences using short probes with a few complementary repeats. Graphical abstract Short oligomeric probes with multiple complementary repeats detect long neurodegenerative targets with high sensitivity and transduce into higher impedance signal.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Expansión de las Repeticiones de ADN , Sondas de Oligonucleótidos/genética , Secuencia de Bases , Técnicas Biosensibles/métodos , Espectroscopía Dieléctrica/métodos , Humanos , Enfermedad de Huntington/genética , ARN/genéticaRESUMEN
DNA substitutions in RNA can probe the importance of A-form structure, 2'-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. Using this approach, we found that Cas9 biochemical activity tolerated significant substitution with DNA nucleotides in the clustered regularly interspaced short palindromic repeat RNA (crRNA). Only minimal RNA content was needed in or near the seed region. Simultaneous substitution at all positions with predicted crRNA-Cas9 2'-hydroxyl contacts had no effect on enzyme activity. The trans-activating crRNA (tracrRNA) also tolerated >50% substitution with DNA. DNA substitutions in the tracrRNA-pairing region of crRNA consistently enhanced cleavage activity while maintaining or improving target specificity. Together, results point to a prominent role for guide:target A-form-like helical structure and a possible regulatory role for the crRNA-tracrRNA pairing motif. A model chimeric crRNA with high activity did not significantly alter RNP assembly or target binding but did reduce Cas9 ribonucleoprotein stability, suggesting effects through conformation or dynamics. Cas9 directed by chimeric RNA-DNA guides may represent a cost-effective synthetic or molecular biology tool for robust and specific DNA cleavage.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Endonucleasas/química , Endonucleasas/genética , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR , Proteínas Asociadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/genética , División del ADN , Endonucleasas/metabolismo , ARN Bacteriano/química , ARN Guía de KinetoplastidaRESUMEN
Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with Flag-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that interactions between functionally important components of RNAi machinery are conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Argonautas/metabolismo , Humanos , Espectrometría de Masas , Unión ProteicaRESUMEN
Expansion of a GGGGCC/CCCCGG repeat sequence in the first intron of the C9ORF72 gene is a leading cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this combined disorder, called c9FTD/ALS, the expansion is bidirectionally transcribed into sense and antisense repeat RNA associated with disease. To better understand the role of C9ORF72 repeat RNA in molecular disease pathology, we determined crystal structures of a [(CCCCGG)3(CCCC)] model antisense repeat RNA to 1.47 Å resolution. The RNA structure was an A-form-like double helix composed of repeating and regularly spaced tandem C:C mismatch pairs that perturbed helical geometry and surface charge. Solution studies revealed a preference for A-form-like helical conformations as the repeat number increased. Results provide a structural starting point for rationalizing the contribution of repeat RNA to c9FTD/ALS molecular disease mechanisms and for developing molecules to target C9ORF72 repeat RNA as potential therapeutics.
Asunto(s)
Disparidad de Par Base/fisiología , Proteínas/química , Proteínas/genética , ARN sin Sentido/química , ARN sin Sentido/genética , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72 , Expansión de las Repeticiones de ADN/fisiología , Demencia Frontotemporal/genética , Humanos , Estructura Secundaria de Proteína , Difracción de Rayos XRESUMEN
Although many long non-coding RNAs (lncRNAs) have been discovered, their function and their association with RNAi factors in the nucleus have remained obscure. Here, we identify RNA transcripts that overlap the cyclooxygenase-2 (COX-2) promoter and contain two adjacent binding sites for an endogenous miRNA, miR-589. We find that miR-589 binds the promoter RNA and activates COX-2 transcription. In addition to miR-589, fully complementary duplex RNAs that target the COX-2 promoter transcript activate COX-2 transcription. Activation by small RNA requires RNAi factors argonaute-2 (AGO2) and GW182, but does not require AGO2-mediated cleavage of the promoter RNA. Instead, the promoter RNA functions as a scaffold. Binding of AGO2 protein/small RNA complexes to the promoter RNA triggers gene activation. Gene looping allows interactions between the promoters of COX-2 and phospholipase A2 (PLA2G4A), an adjacent pro-inflammatory pathway gene that produces arachidonic acid, the substrate for COX-2 protein. miR-589 and fully complementary small RNAs regulate both COX-2 and PLA2G4A gene expression, revealing an unexpected connection between key steps of the eicosanoid signaling pathway. The work demonstrates the potential for RNA to coordinate locus-dependent assembly of related genes to form functional operons through cis-looping.
Asunto(s)
Ciclooxigenasa 2/genética , Fosfolipasas A2 Grupo IV/genética , Regiones Promotoras Genéticas , ARN Pequeño no Traducido/metabolismo , Activación Transcripcional , Proteínas Argonautas/metabolismo , Autoantígenos/metabolismo , Línea Celular Tumoral , Histonas/metabolismo , Humanos , MicroARNs/metabolismo , ARN/biosíntesis , ARN sin Sentido/biosíntesis , Proteínas de Unión al ARN/metabolismoRESUMEN
Huntington's disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal, but reduction of wild-type HTT (wtHTT) is undesirable, as it compromises gene function and potential therapeutic efficacy. One promising allele-selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing (RNA-seq) to monitor the processing precision of a limited set of CAG repeat-targeted small RNAs expressed from multiple scaffold contexts. Small RNA-seq identified expression constructs with high-guide strand 5' processing precision and promising allele-selective inhibition of mutHTT. Transcriptome-wide mRNA-seq also identified an allele-selective small RNA with a favorable off-target profile. These results support continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process.
RESUMEN
Box C/D RNA-protein complexes (RNPs) guide the 2'-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C'/D' RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity.
Asunto(s)
Proteínas Arqueales/química , Proteínas Cromosómicas no Histona/química , Methanococcales/química , Metiltransferasas/química , Ribonucleoproteínas/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Methanococcales/genética , Methanococcales/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
The archaeal L7Ae and eukaryotic 15.5kD protein homologs are members of the L7Ae/15.5kD protein family that characteristically recognize K-turn motifs found in both archaeal and eukaryotic RNAs. In Archaea, the L7Ae protein uniquely binds the K-loop motif found in box C/D and H/ACA sRNAs, whereas the eukaryotic 15.5kD homolog is unable to recognize this variant K-turn RNA. Comparative sequence and structural analyses, coupled with amino acid replacement experiments, have demonstrated that five amino acids enable the archaeal L7Ae core protein to recognize and bind the K-loop motif. These signature residues are highly conserved in the archaeal L7Ae and eukaryotic 15.5kD homologs, but differ between the two domains of life. Interestingly, loss of K-loop binding by archaeal L7Ae does not disrupt C'/D' RNP formation or RNA-guided nucleotide modification. L7Ae is still incorporated into the C'/D' RNP despite its inability to bind the K-loop, thus indicating the importance of protein-protein interactions for RNP assembly and function. Finally, these five signature amino acids are distinct for each of the L7Ae/L30 family members, suggesting an evolutionary continuum of these RNA-binding proteins for recognition of the various K-turn motifs contained in their cognate RNAs.
Asunto(s)
Conformación de Ácido Nucleico , ARN de Archaea/química , ARN de Archaea/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Archaea/genética , Archaea/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Secuencia Conservada , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica/genética , Ribonucleoproteínas/genética , Homología de Secuencia de AminoácidoRESUMEN
Transcriptome studies reveal many noncoding transcripts overlapping 3' gene termini. The function of these transcripts is unknown. Here we have characterized transcription at the progesterone receptor (PR) locus and identified noncoding transcripts that overlap the 3' end of the gene. Small RNAs complementary to sequences beyond the 3' terminus of PR mRNA modulated expression of PR, recruited argonaute 2 to a 3' noncoding transcript, altered occupancy of RNA polymerase II, induced chromatin changes at the PR promoter and affected responses to physiological stimuli. We found that the promoter and 3' terminal regions of the PR locus are in close proximity, providing a potential mechanism for RNA-mediated control of transcription over long genomic distances. These results extend the potential for small RNAs to regulate transcription to target sequences beyond the 3' termini of mRNA.
Asunto(s)
Regiones no Traducidas 3'/genética , Regulación de la Expresión Génica/fisiología , ARN Citoplasmático Pequeño/fisiología , Regiones no Traducidas 3'/fisiología , Regiones no Traducidas 5'/genética , Proteína BRCA1/biosíntesis , Proteína BRCA1/genética , Línea Celular , Cromatina/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/genética , Perfilación de la Expresión Génica , Marcación de Gen , Humanos , Conformación Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Short hairpin RNAs, or short hairpin RNAs (shRNAs), are a proven tool for gene knockdown and a promising therapeutic approach for suppression of disease-associated genes. The efficient preparation of shRNA-expressing vectors can sometimes become a bottleneck due to the complexity of shRNA hairpin sequence and structure, especially for repetitive or high GC-content targets. Here, we present improved shRNA cloning and validation methods that enabled efficient and rapid cloning of several shRNAs targeting disease-associated repeat expansions, including GGGGCC, CAG, CTG, CCTG, and CGG into modified pLKO.1 vectors. Improvements included shRNA insert design and preparation, recombination-based cloning, and sequencing-based validation that included Sanger and nanopore long-read sequencing. This improved method should enable practical, efficient cloning of nearly any shRNA sequence.
Asunto(s)
Vectores Genéticos , Clonación Molecular , Técnicas de Silenciamiento del Gen , Vectores Genéticos/genética , ARN Interferente Pequeño/genéticaRESUMEN
Oligonucleotides and their derivatives are a proven chemical strategy for modulating gene expression. However, their negative charge remains a challenge for delivery and target recognition inside cells. Here we show that oligonucleotide-oligospermine conjugates (Zip nucleic acids or ZNAs) can help overcome these shortcomings by serving as effective antisense and antigene agents. Conjugates containing DNA and locked nucleic acid (LNA) oligonucleotides are active, and oligospermine conjugation facilitates carrier-free cell uptake at nanomolar concentrations. Conjugates targeting the CAG triplet repeat within huntingtin (HTT) mRNA selectively inhibit expression of the mutant huntingtin protein. Conjugates targeting the promoter of the progesterone receptor (PR) function as antigene agents to block PR expression. These observations support further investigation of ZNA conjugates as gene silencing agents.