RESUMO
Repeat-associated non-AUG (RAN) translation was discovered in 2011 in spinocerebellar ataxia type 8 (SCA8) and myotonic dystrophy type 1 (DM1). This non-canonical form of translation occurs in all reading frames from both coding and non-coding regions of sense and antisense transcripts carrying expansions of trinucleotide to hexanucleotide repeat sequences. RAN translation has since been reported in 7 of the 53 known microsatellite expansion disorders which mainly present with neurodegenerative features. RAN translation leads to the biosynthesis of low-complexity polymeric repeat proteins with aggregating and cytotoxic properties. However, the molecular mechanisms and protein factors involved in assembling functional ribosomes in absence of canonical AUG start codons remain poorly characterised while secondary repeat RNA structures play key roles in initiating RAN translation. Here, we briefly review the repeat expansion disorders, their complex pathogenesis and the mechanisms of physiological translation initiation together with the known factors involved in RAN translation. Finally, we discuss research challenges surrounding the understanding of pathogenesis and future directions that may provide opportunities for the development of novel therapeutic approaches for this group of incurable neurodegenerative diseases.
Assuntos
Códon de Iniciação/genética , Repetições de Microssatélites/genética , Doenças do Sistema Nervoso/genética , Biossíntese de Proteínas/genética , Expansão das Repetições de Trinucleotídeos/genética , Ataxinas/genética , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Degenerações Espinocerebelares/genéticaRESUMO
In contrast to -1 programmed ribosomal frameshifting (PRF) stimulation by an RNA pseudoknot downstream of frameshifting sites, a refolding upstream RNA hairpin juxtaposing the frameshifting sites attenuates -1 PRF in human cells and stimulates +1 frameshifting in yeast. This eukaryotic functional mimicry of the internal Shine-Dalgarno (SD) sequence-mediated duplex was confirmed directly in the 70S translation system, indicating that both frameshifting regulation activities of upstream hairpin are conserved between 70S and 80S ribosomes. Unexpectedly, a downstream pseudoknot also possessed two opposing hungry codon-mediated frameshifting regulation activities: attenuation of +1 frameshifting and stimulation of a non-canonical -1 frameshifting within the +1 frameshift-prone CUUUGA frameshifting site in the absence of release factor 2 (RF2) in vitro. However, the -1 frameshifting activity of the downstream pseudoknot is not coupled with its +1 frameshifting attenuation ability. Similarly, the +1 frameshifting activity of the upstream hairpin is not required for its -1 frameshifting attenuation function Thus, each of the mRNA duplexes flanking the two ends of a ribosomal mRNA-binding channel possesses two functions in bi-directional ribosomal frameshifting regulation: frameshifting stimulation and counteracting the frameshifting activity of each other.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Ácidos Nucleicos Heteroduplexes/metabolismo , RNA Mensageiro/metabolismo , Sequência de Bases , Códon/genética , Fases de Leitura Aberta/genética , Fatores de Terminação de Peptídeos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/química , Ribossomos/metabolismoRESUMO
Frameshifting is an essential process that regulates protein synthesis in many viruses. The ribosome may slip backward when encountering a frameshift motif on the messenger RNA, which usually contains a pseudoknot structure involving tertiary base pair interactions. Due to the lack of detailed molecular explanations, previous studies investigating which features of the pseudoknot are important to stimulate frameshifting have presented diverse conclusions. Here we constructed a bimolecular pseudoknot to dissect the interior tertiary base pairs and used single-molecule approaches to assess the structure targeted by ribosomes. We found that the first ribosome target stem was resistant to unwinding when the neighboring loop was confined along the stem; such constrained conformation was dependent on the presence of consecutive adenosines in this loop. Mutations that disrupted the distal base triples abolished all remaining tertiary base pairs. Changes in frameshifting efficiency correlated with the stem unwinding resistance. Our results demonstrate that various tertiary base pairs are coordinated inside a highly efficient frameshift-stimulating RNA pseudoknot and suggest a mechanism by which mechanical resistance of the pseudoknot may persistently act on translocating ribosomes.
Assuntos
Pareamento de Bases , Mudança da Fase de Leitura do Gene Ribossômico/fisiologia , Conformação de Ácido Nucleico , RNA Mensageiro/química , Ribossomos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química , Pinças Ópticas , RNA Mensageiro/genética , Fases de Leitura , Especificidade por SubstratoRESUMO
Metabolite-responsive RNA pseudoknots derived from prokaryotic riboswitches have been shown to stimulate -1 programmed ribosomal frameshifting (PRF), suggesting -1 PRF as a promising gene expression platform to extend riboswitch applications in higher eukaryotes. However, its general application has been hampered by difficulty in identifying a specific ligand-responsive pseudoknot that also functions as a ligand-dependent -1 PRF stimulator. We addressed this problem by using the -1 PRF stimulation pseudoknot of SARS-CoV (SARS-PK) to build a ligand-dependent -1 PRF stimulator. In particular, the extra stem of SARS-PK was replaced by an RNA aptamer of theophylline and designed to couple theophylline binding with the stimulation of -1 PRF. Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent -1 PRF stimulation activity in a stable human 293T cell-line. Thus, RNA-ligand interaction repertoire provided by in vitro selection becomes accessible to ligand-specific -1 PRF stimulator engineering using SARS-PK as the scaffold for synthetic biology application.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Ligantes , RNA Viral/química , Riboswitch , Animais , Sequência de Bases , Linhagem Celular , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Humanos , Sequências Repetidas Invertidas , Mamíferos , Modelos Biológicos , Conformação de Ácido Nucleico , RNA Viral/genética , Riboswitch/efeitos dos fármacos , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Biologia Sintética , Teofilina/química , Teofilina/farmacologiaRESUMO
Viral -1 programmed ribosomal frameshifting (PRF) as a potential antiviral target has attracted interest because many human viral pathogens, including human immunodeficiency virus (HIV) and coronaviruses, rely on -1 PRF for optimal propagation. Efficient eukaryotic -1 PRF requires an optimally placed stimulator structure downstream of the frameshifting site and different strategies targeting viral -1 PRF stimulators have been developed. However, accessing particular -1 PRF stimulator information represents a bottle-neck in combating the emerging epidemic viral pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV). Recently, an RNA hairpin upstream of frameshifting site was shown to act as a cis-element to attenuate -1 PRF with mechanism unknown. Here, we show that an upstream duplex formed in-trans, by annealing an antisense to its complementary mRNA sequence upstream of frameshifting site, can replace an upstream hairpin to attenuate -1 PRF efficiently. This finding indicates that the formation of a proximal upstream duplex is the main determining factor responsible for -1 PRF attenuation and provides mechanistic insight. Additionally, the antisense-mediated upstream duplex approach downregulates -1 PRF stimulated by distinct -1 PRF stimulators, including those of MERS-CoV, suggesting its general application potential as a robust means to evaluating viral -1 PRF inhibition as soon as the sequence information of an emerging human coronavirus is available.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , RNA Viral/genética , Linhagem Celular , DNA/genética , DNA/metabolismo , DNA Antissenso/genética , DNA Antissenso/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Sequências Repetidas Invertidas , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , RNA Mensageiro/genética , RNA Viral/química , RNA Viral/metabolismo , Ribossomos/metabolismoRESUMO
Distinct translational initiation mechanisms between prokaryotes and eukaryotes limit the exploitation of prokaryotic riboswitch repertoire for regulatory RNA circuit construction in mammalian application. Here, we explored programmed ribosomal frameshifting (PRF) as the regulatory gene expression platform for engineered ligand-responsive RNA devices in higher eukaryotes. Regulation was enabled by designed ligand-dependent conformational rearrangements of the two cis-acting RNA motifs of opposite activity in -1 PRF. Particularly, RNA elements responsive to trans-acting ligands can be tailored to modify co-translational RNA refolding dynamics of a hairpin upstream of frameshifting site to achieve reversible and adjustable -1 PRF attenuating activity. Combined with a ligand-responsive stimulator, synthetic RNA devices for synergetic translational-elongation control of gene expression can be constructed. Due to the similarity between co-transcriptional RNA hairpin folding and co-translational RNA hairpin refolding, the RNA-responsive ligand repertoire provided in prokaryotic systems thus becomes accessible to gene-regulatory circuit construction for synthetic biology application in mammalian cells.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Riboswitch , Regiões Terminadoras Genéticas , Proteínas de Bactérias/metabolismo , Células HEK293 , Humanos , Ligantes , RNA/metabolismo , Dobramento de RNA , Proteínas de Ligação a RNA/metabolismo , S-Adenosil-Homocisteína/metabolismo , Teofilina/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3' direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) -1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate-1 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for-1 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing-1 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study's findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation.
Assuntos
Pareamento de Bases , Mudança da Fase de Leitura do Gene Ribossômico , Sequências Repetidas Invertidas , RNA Viral/química , RNA Viral/genética , Sequência de Bases , Motivos de Nucleotídeos , Estabilidade de RNA , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Saccharomyces cerevisiae/genéticaRESUMO
Specific recognition of metabolites by functional RNA motifs within mRNAs has emerged as a crucial regulatory strategy for feedback control of biochemical reactions. Such riboswitches have been demonstrated to regulate different gene expression processes, including transcriptional termination and translational initiation in prokaryotic cells, as well as splicing in eukaryotic cells. The regulatory process is usually mediated by modulating the accessibility of specific sequence information of the expression platforms via metabolite-induced RNA conformational rearrangement. In eukaryotic systems, viral and the more limited number of cellular decoding -1 programmed ribosomal frameshifting (PRF) are commonly promoted by a 3' mRNA pseudoknot. In addition, such -1 PRF is generally constitutive rather than being regulatory, and usually results in a fixed ratio of products. We report here an RNA pseudoknot capable of stimulating -1 PRF whose efficiency can be tuned in response to the concentration of S-adenosylhomocysteine (SAH), and the improvement of its frameshifting efficiency by RNA engineering. In addition to providing an alternative approach for small-molecule regulation of gene expression in eukaryotic cells, such a metabolite-responsive pseudoknot suggests a plausible mechanism for metabolite-driven translational regulation of gene expression in eukaryotic systems.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA/genética , Adenosil-Homocisteinase/metabolismo , Sequência de Bases , Técnicas de Cultura de Células , Linhagem Celular , Expressão Gênica , HIV-1/genética , Humanos , Rim/embriologia , Luciferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA/química , RNA/metabolismo , RNA Catalítico/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
An efficient -1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote -1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several -1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of -1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the -1 PRF efficiency of this stimulator can be modulated by sequential removal of base-triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem-loop interactions and their relative positions to the helical junction play crucial roles for the -1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate -1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate -1 PRF. Furthermore, it suggests that -1 PRF, induced by an in-trans RNA via specific base-triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , RNA não Traduzido/química , RNA/química , Telomerase/química , Humanos , Mutação , Conformação de Ácido Nucleico , RNA Mensageiro/químicaRESUMO
Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Conformação de Ácido Nucleico , Estabilidade de RNA , RNA Mensageiro/químicaRESUMO
The -1 ribosomal frameshifting requires the existence of an in cis RNA slippery sequence and is promoted by a downstream stimulator RNA. An atypical RNA pseudoknot with an extra stem formed by complementary sequences within loop 2 of an H-type pseudoknot is characterized in the severe acute respiratory syndrome coronavirus (SARS CoV) genome. This pseudoknot can serve as an efficient stimulator for -1 frameshifting in vitro. Mutational analysis of the extra stem suggests frameshift efficiency can be modulated via manipulation of the secondary structure within the loop 2 of an infectious bronchitis virus-type pseudoknot. More importantly, an upstream RNA sequence separated by a linker 5' to the slippery site is also identified to be capable of modulating the -1 frameshift efficiency. RNA sequence containing this attenuation element can downregulate -1 frameshifting promoted by an atypical pseudoknot of SARS CoV and two other pseudoknot stimulators. Furthermore, frameshift efficiency can be reduced to half in the presence of the attenuation signal in vivo. Therefore, this in cis RNA attenuator represents a novel negative determinant of general importance for the regulation of -1 frameshift efficiency, and is thus a potential antiviral target.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Regulação Viral da Expressão Gênica , RNA Viral/química , Sequências Reguladoras de Ácido Ribonucleico , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Sequência de Bases , Regulação para Baixo , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
The double-stranded RNA-binding motif (dsRBM) is an alphabetabetabetaalpha fold with a well-characterized function to bind structured RNA molecules. This motif is widely distributed in eukaryotic proteins, as well as in proteins from bacteria and viruses. dsRBM-containing proteins are involved in processes ranging from RNA editing to protein phosphorylation in translational control and contain a variable number of dsRBM domains. The structural work of the past five years has identified a common mode of RNA target recognition by dsRBMs and dissected this recognition into two functionally separated interaction modes. The first involves the recognition of specific moieties of the RNA A-form helix by two protein loops, while the second is based on the interaction between structural elements flanking the RNA duplex with the first helix of the dsRBM. The latter interaction can be tuned by other protein elements. Recent work has made clear that dsRBMs can also recognize non-RNA targets (proteins and DNA), and act in combination with other dsRBMs and non-dsRBM motifs to play a regulatory role in catalytic processes. The elucidation of functional networks coordinated by dsRBM folds will require information on the precise functional relationship between different dsRBMs and a clarification of the principles underlying dsRBM-protein recognition.
Assuntos
Sequência de Aminoácidos , Conformação Proteica , RNA de Cadeia Dupla/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Alinhamento de SequênciaRESUMO
Structure-based mutagenesis was used to probe the binding surface for the activation domain of sterol-responsive element binding protein (SREBP) in the KIX domain of CREB binding protein. A set of conserved residues scattering in the alpha2 helix and the extended C-terminal region of alpha 3 helix in the KIX domain including two arginines previously characterized as a hot spot for cofactor-mediated methylation was shown to be crucial for SREBP-KIX interaction, and was not essential for phosphorylated KID recognition. Therefore, our results suggest the existence of a SREBP binding site formed by positively charged residues in the C-terminal part of the extended alpha 3 helix of the KIX domain distinct from the previously identified phosphorylated KID binding site.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteína de Ligação a CREB , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/química , Oligonucleotídeos/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Proteína de Ligação a Elemento Regulador de Esterol 1 , Transativadores/químicaRESUMO
The double-stranded RNA-binding motif (dsRBM) is a widely distributed motif frequently found within proteins with sequence non-specific RNA duplex-binding activity. In addition to the binding of double-stranded RNA, some dsRBMs also participate in complex formation via protein-protein interactions. Interestingly, a lot of proteins containing multiple dsRBMs have only some of their dsRBMs with the expected RNA duplex-binding competency proven, while the functions of the other dsRBMs remain unknown. We show here that the dsRBM1 of RNA helicase A (RHA) can cooperate with a C-terminal domain of proline-rich content to gain novel nucleic acid-binding activities. This integrated nucleic acid-binding module is capable of associating with the consensus sequences of the constitutive transport element (CTE) RNA of type D retrovirus against RNA duplex competitors. Remarkably, binding activity for double-stranded DNA corresponding to the consensus sequences of the cyclic-AMP responsive element also resides within this composite nucleic acid binder. It thus suggests that the dsRBM fold can be used as a platform for the building of a ligand binding module capable of non-RNA macromolecule binding with an accessory sequence, and functional assessment for a newly identified protein containing dsRBM fold should be more cautious.