RESUMO
Human Argonaute 2 (hAgo2) constitutes the functional core of the RNA interference pathway. Guide RNAs direct hAgo2 to target mRNAs, which ultimately leads to hAgo2-mediated mRNA degradation or translational inhibition. Here, we combine site-specifically labeled hAgo2 with time-resolved single-molecule FRET measurements to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observe dynamic anchoring and release of the guide's 3'-end from the PAZ domain during the stepwise target loading process even with a fully complementary target. We find differences in structure and dynamic behavior between partially and fully paired canonical hAgo2-guide/target complexes and the miRNA processing complex formed by hAgo2 and pre-miRNA451. Furthermore, we detect a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. Taken together, our results show how the conformational flexibility of hAgo2-RNA complexes determines function and the fate of the ribonucleoprotein particle.
Assuntos
Proteínas Argonautas , MicroRNAs , Proteínas Argonautas/química , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação Molecular , Estabilidade de RNA , RNA MensageiroRESUMO
CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Clivagem do DNA , DNA/metabolismo , Streptococcus pyogenes/enzimologia , Algoritmos , Proteína 9 Associada à CRISPR/genética , DNA/genética , Estabilidade Enzimática/genética , Magnetismo , Mutação , Pinças Ópticas , Ligação Proteica , Estruturas R-Loop/genética , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/genéticaRESUMO
The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Proteínas de Bactérias/genética , Pareamento Incorreto de Bases , Proteínas Associadas a CRISPR/genética , Endodesoxirribonucleases/genética , Francisella/enzimologia , Mutação , Conformação ProteicaRESUMO
Many biophysical techniques that are available to study the structure, function and dynamics of cellular constituents require modification of the target molecules. Site-specific labelling of a protein is of particular interest for fluorescence-based single-molecule measurements including single-molecule FRET or super-resolution microscopy. The labelling procedure should be highly specific but minimally invasive to preserve sensitive biomolecules. The modern molecular engineering toolkit provides elegant solutions to achieve the site-specific modification of a protein of interest often necessitating the incorporation of an unnatural amino acid to introduce a unique reactive moiety. The Amber suppression strategy allows the site-specific incorporation of unnatural amino acids into a protein of interest. Recently, this approach has been transferred to the mammalian expression system. Here, we demonstrate how the combination of unnatural amino acid incorporation paired with current bioorthogonal labelling strategies allow the site-specific engineering of fluorescent dyes into proteins produced in the cellular environment of a human cell. We describe in detail which parameters are important to ensure efficient incorporation of unnatural amino acids into a target protein in human expression systems. We furthermore outline purification and bioorthogonal labelling strategies that allow fast protein preparation and labelling of the modified protein. This way, the complete eukaryotic proteome becomes available for single-molecule fluorescence assays.
RESUMO
Fluorescence resonance energy transfer (FRET) has been instrumental in determining the structure and dynamics of biomolecules but distances above 8 nanometers are not accessible. However, with the advent and rapid development of super-resolution (SR) microscopy, distances between two fluorescent dyes below 20 nanometers can be resolved, which hitherto has been inaccessible for fluorescence microscopy approaches due to the limited resolving power of an optical imaging system that is determined by the fundamental laws of light diffraction (referred to as the diffraction limit). Therefore, the question arises whether SR microscopy can ultimately close the resolution gap between FRET and the diffraction limit and whether SR microscopy can be employed for the structural interrogation of proteins in the sub-20 nm range? Here, we show that the combination of DNA nanotechnology and single-molecule biochemistry allows the first step towards the investigation of the structural organization of a protein via SR microscopy. Limiting factors and possible future directions for the full implementation of SR microscopy as a structural tool are discussed.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência , Nanotecnologia , DNA , Corantes FluorescentesRESUMO
Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single-molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site-specific coupling of organic dyes into native, non-recombinant mammalian proteins is lacking. We address this issue showing the site-specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post-translationally modified proteins on the single-molecule level, among them the hitherto inaccessible human Argonauteâ 2.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas/química , Cromatografia de Afinidade , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Proteínas/isolamento & purificação , ProteomaRESUMO
Double-stranded RNA-binding domains (dsRBDs) are commonly found in modular proteins that interact with RNA. Two varieties of dsRBD exist: canonical Type A dsRBDs interact with dsRNA, while non-canonical Type B dsRBDs lack RNA-binding residues and instead interact with other proteins. In higher eukaryotes, the microRNA biogenesis enzyme Dicer forms a 1:1 association with a dsRNA-binding protein (dsRBP). Human Dicer associates with HIV TAR RNA-binding protein (TRBP) or protein activator of PKR (PACT), while Drosophila Dicer-1 associates with Loquacious (Loqs). In each case, the interaction involves a region of the protein that contains a Type B dsRBD. All three dsRBPs are reported to homodimerize, with the Dicer-binding region implicated in self-association. We report that these dsRBD homodimers display structural asymmetry and that this unusual self-association mechanism is conserved from flies to humans. We show that the core dsRBD is sufficient for homodimerization and that mutation of a conserved leucine residue abolishes self-association. We attribute differences in the self-association properties of Loqs, TRBP and PACT to divergence of the composition of the homodimerization interface. Modifications that make TRBP more like PACT enhance self-association. These data are examined in the context of miRNA biogenesis and the protein/protein interaction properties of Type B dsRBDs.
Assuntos
Proteínas de Ligação a RNA/química , Proteínas de Drosophila , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Multimerização Proteica , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 5' leader and 3' trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading.
Assuntos
Autoantígenos/metabolismo , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência de Isoleucina/metabolismo , Ribonucleoproteínas/metabolismo , Células A549 , Proteínas Argonautas/metabolismo , Autoantígenos/genética , Sítios de Ligação , RNA Helicases DEAD-box/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Humanos , Carioferinas/metabolismo , Células MCF-7 , MicroRNAs/genética , Conformação de Ácido Nucleico , Ligação Proteica , Interferência de RNA , RNA Polimerase III/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , RNA de Transferência de Isoleucina/química , RNA de Transferência de Isoleucina/genética , RNA Viral/genética , RNA Viral/metabolismo , Ribonuclease III/metabolismo , Ribonucleoproteínas/genética , Relação Estrutura-Atividade , Transfecção , Antígeno SS-BRESUMO
In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (dmDcr1) to facilitate miRNA processing. Here, we have solved the structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs and define specific structural elements that interact with dmDcr1. In addition, we show that the linker preceding dsRBD3 contributes significantly to dmDcr1 binding. Furthermore, our structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate dmDcr1 interaction. Loqs, however, binds to dmDcr1 as a monomer using the identified dimerization surface, which suggests that Loqs might form dimers under conditions where dmDcr1 is absent or not accessible. Since critical sequence elements are conserved, we suggest that dimerization might be a general feature of dsRBD proteins in gene silencing.
Assuntos
Drosophila melanogaster/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/fisiologia , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Dimerização , Proteínas de Drosophila , Inativação Gênica , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , Espectrometria de FluorescênciaRESUMO
TRIM-NHL proteins are conserved among metazoans and control cell fate decisions in various stem cell linages. The Drosophila TRIM-NHL protein Brain tumor (Brat) directs differentiation of neuronal stem cells by suppressing self-renewal factors. Brat is an RNA-binding protein and functions as a translational repressor. However, it is unknown which RNAs Brat regulates and how RNA-binding specificity is achieved. Using RNA immunoprecipitation and RNAcompete, we identify Brat-bound mRNAs in Drosophila embryos and define consensus binding motifs for Brat as well as a number of additional TRIM-NHL proteins, indicating that TRIM-NHL proteins are conserved, sequence-specific RNA-binding proteins. We demonstrate that Brat-mediated repression and direct RNA-binding depend on the identified motif and show that binding of the localization factor Miranda to the Brat-NHL domain inhibits Brat activity. Finally, to unravel the sequence specificity of the NHL domain, we crystallize the Brat-NHL domain in complex with RNA and present a high-resolution protein-RNA structure of this fold.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Drosophila/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Dados de Sequência Molecular , Ligação Proteica , RNA Mensageiro/metabolismoRESUMO
During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.