RESUMEN
Non-CpG PS-ASOs can activate the innate immune system, leading to undesired outcomes. This response can vary-in part-as a function of 2'modifications and sequence. Here we investigated the molecular steps involved in the varied effects of PS-ASOs on the innate immune system. We found that pro-inflammatory PS-ASOs require TLR9 signaling based on the experimental systems used. However, the innate immunity of PS-ASOs does not correlate with their binding affinity with TLR9. Furthermore, the innate immune responses of pro-inflammatory PS-ASOs were reduced by coincubation with non-inflammatory PS-ASOs, suggesting that both pro-inflammatory and non-inflammatory PS-ASOs can interact with TLR9. We show that the kinetics of the PS-ASO innate immune responses can vary, which we speculate may be due to the existence of alternative PS-ASO binding sites on TLR9, leading to full, partial, or no activation of the pathway. In addition, we found that several extracellular proteins, including HMGB1, S100A8 and HRG, enhance the innate immune responses of PS-ASOs. Reduction of the binding affinity by reducing the PS content of PS-ASOs decreased innate immune responses, suggesting that PS-ASO-protein complexes may be sensed by TLR9. These findings thus provide critical information concerning how PS-ASOs can interact with and activate TLR9.
Asunto(s)
Inmunidad Innata , Oligonucleótidos Antisentido , Oligonucleótidos Fosforotioatos , Receptor Toll-Like 9 , Calgranulina A , Endocitosis , Proteína HMGB1 , Humanos , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Proteínas , Receptor Toll-Like 9/metabolismoRESUMEN
We recently found that toxic PS-ASOs can cause P54nrb and PSF nucleolar mislocalization in an RNase H1-dependent manner. To better understand the underlying mechanisms of these observations, here we utilize different biochemical approaches to demonstrate that PS-ASO binding can alter the conformations of the bound proteins, as illustrated using recombinant RNase H1, P54nrb, PSF proteins and various isolated domains. While, in general, binding of PS-ASOs or ASO/RNA duplexes stabilizes the conformations of these proteins, PS-ASO binding may also cause the unfolding of RNase H1, including both the hybrid binding domain and the catalytic domain. The extent of conformational change correlates with the binding affinity of PS-ASOs to the proteins. Consequently, PS-ASO binding to RNase H1 induces the interaction of RNase H1 with P54nrb or PSF in a 2'-modification and sequence dependent manner, and toxic PS-ASOs tend to induce more interactions than non-toxic PS-ASOs. PS-ASO binding also enhances the interaction between P54nrb and PSF. However, the interaction between RNase H1 and P32 protein can be disrupted upon binding of PS-ASOs. Together, these results suggest that stronger binding of PS-ASOs can cause greater conformational changes of the bound proteins, subsequently affecting protein-protein interactions. These observations thus provide deeper understanding of the molecular basis of PS-ASO-induced protein mislocalization or degradation observed in cells and advance our understanding of why some PS-ASOs are cytotoxic.
Asunto(s)
Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Ribonucleasa H/metabolismo , Línea Celular , Quimotripsina , Humanos , Proteínas Nucleares/metabolismo , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/química , Unión Proteica , Conformación Proteica , Señales de Clasificación de Proteína , ARN/metabolismo , Ribonucleasa H/químicaRESUMEN
The PS modification enhances the nuclease stability and protein binding properties of gapmer antisense oligonucleotides (ASOs) and is one of very few modifications that support RNaseH1 activity. We evaluated the effect of introducing stereorandom and chiral mesyl-phosphoramidate (MsPA) linkages in the DNA gap and flanks of gapmer PS ASOs and characterized the effect of these linkages on RNA-binding, nuclease stability, protein binding, pro-inflammatory profile, antisense activity and toxicity in cells and in mice. We show that all PS linkages in a gapmer ASO can be replaced with MsPA without compromising chemical stability and RNA binding affinity but these designs reduced activity. However, replacing up to 5 PS in the gap with MsPA was well tolerated and replacing specific PS linkages at appropriate locations was able to greatly reduce both immune stimulation and cytotoxicity. The improved nuclease stability of MsPA over PS translated to significant improvement in the duration of ASO action in mice which was comparable to that of enhanced stabilized siRNA designs. Our work highlights the combination of PS and MsPA linkages as a next generation chemical platform for identifying ASO drugs with improved potency and therapeutic index, reduced pro-inflammatory effects and extended duration of effect.
Asunto(s)
Oligonucleótidos Antisentido/síntesis química , Índice Terapéutico de los Medicamentos , Animales , Células HEK293 , Células HeLa , Humanos , Hígado/metabolismo , Masculino , Mesilatos/química , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Antisentido/toxicidad , Fosforamidas/química , Unión Proteica , Distribución TisularRESUMEN
Antisense oligonucleotides (ASOs) interact with target RNAs via hybridization to modulate gene expression through different mechanisms. ASO therapeutics are chemically modified and include phosphorothioate (PS) backbone modifications and different ribose and base modifications to improve pharmacological properties. Modified PS ASOs display better binding affinity to the target RNAs and increased binding to proteins. Moreover, PS ASO protein interactions can affect many aspects of their performance, including distribution and tissue delivery, cellular uptake, intracellular trafficking, potency and toxicity. In this review, we summarize recent progress in understanding PS ASO protein interactions, highlighting the proteins with which PS ASOs interact, the influence of PS ASO protein interactions on ASO performance, and the structure activity relationships of PS ASO modification and protein interactions. A detailed understanding of these interactions can aid in the design of safer and more potent ASO drugs, as illustrated by recent findings that altering ASO chemical modifications dramatically improves therapeutic index.
Asunto(s)
Oligonucleótidos Fosforotioatos/química , Proteínas/química , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Espacio Intracelular/química , Espacio Intracelular/metabolismo , Ligandos , Oligonucleótidos Fosforotioatos/metabolismo , Oligonucleótidos Fosforotioatos/farmacología , Oligonucleótidos Fosforotioatos/toxicidad , Unión Proteica , Dominios Proteicos , Proteínas/metabolismo , Proteínas/toxicidad , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Relación Estructura-Actividad , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Release of phosphorothioate antisense oligonucleotides (PS-ASOs) from late endosomes (LEs) is a rate-limiting step and a poorly defined process for productive intracellular ASO drug delivery. Here, we examined the role of Golgi-endosome transport, specifically M6PR shuttling mediated by GCC2, in PS-ASO trafficking and activity. We found that reduction in cellular levels of GCC2 or M6PR impaired PS-ASO release from endosomes and decreased PS-ASO activity in human cells. GCC2 relocated to LEs upon PS-ASO treatment, and M6PR also co-localized with PS-ASOs in LEs or on LE membranes. These proteins act through the same pathway to influence PS-ASO activity, with GCC2 action preceding that of M6PR. Our data indicate that M6PR binds PS-ASOs and facilitates their vesicular escape. The co-localization of M6PR and of GCC2 with ASOs is influenced by the PS modifications, which have been shown to enhance the affinity of ASOs for proteins, suggesting that localization of these proteins to LEs is mediated by ASO-protein interactions. Reduction of M6PR levels also decreased PS-ASO activity in mouse cells and in livers of mice treated subcutaneously with PS-ASO, indicating a conserved mechanism. Together, these results demonstrate that the transport machinery between LE and Golgi facilitates PS-ASO release.
Asunto(s)
Endosomas/genética , Proteínas de la Matriz de Golgi/genética , Oligonucleótidos Antisentido/genética , Receptor IGF Tipo 2/genética , Animales , Endocitosis/genética , Endosomas/metabolismo , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Proteínas de la Matriz de Golgi/metabolismo , Células HeLa , Humanos , Ratones , Oligonucleótidos Fosforotioatos/genética , Transporte de Proteínas/genética , Receptor IGF Tipo 2/metabolismoRESUMEN
The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein-protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO-protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa H/metabolismo , Dominio Catalítico , Nucléolo Celular/metabolismo , Supervivencia Celular , Proteínas de Unión al ADN/química , Células HEK293 , Células HeLa , Humanos , Cinética , Unión Proteica , Proteínas de Unión al ARN/química , Ribonucleasa H/químicaRESUMEN
Antisense technology can reduce gene expression via the RNase H1 or RISC pathways and can increase gene expression through modulation of splicing or translation. Here, we demonstrate that antisense oligonucleotides (ASOs) can reduce mRNA levels by acting through the no-go decay pathway. Phosphorothioate ASOs fully modified with 2'-O-methoxyethyl decreased mRNA levels when targeted to coding regions of mRNAs in a translation-dependent, RNase H1-independent manner. The ASOs that activated this decay pathway hybridized near the 3' end of the coding regions. Although some ASOs induced nonsense-mediated decay, others reduced mRNA levels through the no-go decay pathway, since depletion of PELO/HBS1L, proteins required for no-go decay pathway activity, decreased the activities of these ASOs. ASO length and chemical modification influenced the efficacy of these reagents. This non-gapmer ASO-induced mRNA reduction was observed for different transcripts and in different cell lines. Thus, our study identifies a new mechanism by which mRNAs can be degraded using ASOs, adding a new antisense approach to modulation of gene expression. It also helps explain why some fully modified ASOs cause RNA target to be reduced despite being unable to serve as substrates for RNase H1.
Asunto(s)
Oligonucleótidos Antisentido/farmacocinética , Oligonucleótidos Fosforotioatos/farmacología , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Endonucleasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Calor , Ratones , Proteínas Nucleares/metabolismo , Desnaturalización de Ácido Nucleico , Fosfoproteínas/genética , Biosíntesis de Proteínas , Interferencia de ARN , Empalme del ARN , ARN Interferente Pequeño/farmacología , Proteínas de Unión al ARN/genética , NucleolinaRESUMEN
The activity of PS-ASOs is strongly influenced by association with both inter- and intracellular proteins. The sequence, chemical nature, and structure of the ASO can have profound influences on the interaction of PS-ASOs with specific proteins. A more thorough understanding of how these pharmacological agents interact with various proteins and how chemical modifications, sequence, and structure influence interactions with proteins is needed to inform future ASO design efforts. To better understand the chemistry of PS-ASO interactions, we have focused on human positive cofactor 4 (PC4). Although several studies have investigated the in vitro binding properties of PC4 with endogenous nucleic acids, little is known about the chemistry of interaction of PS-ASOs with this protein. Here we examine in detail the impact of ASO backbone chemistry, 2'-modifications, and buffer environment on the binding affinity of PC4. In addition, using site-directed mutagenesis, we identify those amino acids that are specifically required for ASO binding interactions, and by substitution of abasic nucleotides we identify the positions on the ASO that most strongly influence affinity for PC4. Finally, to confirm that the interactions observed in vitro are biologically relevant, we use a recently developed complementation reporter system to evaluate the kinetics and subcellular localization of the interaction of ASO and PC4 in live cells.
Asunto(s)
Proteínas de Unión al ADN/química , Oligonucleótidos Antisentido/química , Factores de Transcripción/química , Células HEK293 , Células HeLa , Humanos , CinéticaRESUMEN
Recent progress in understanding phosphorothioate antisense oligonucleotide (PS-ASO) interactions with proteins has revealed that proteins play deterministic roles in the absorption, distribution, cellular uptake, subcellular distribution, molecular mechanisms of action, and toxicity of PS-ASOs. Similarly, such interactions can alter the fates of many intracellular proteins. These and other advances have opened new avenues for the medicinal chemistry of PS-ASOs and research on all elements of the molecular pharmacology of these molecules. These advances have recently been reviewed. In this Perspective article, we summarize some of those learnings, the general principles that have emerged, and a few of the exciting new questions that can now be addressed.
Asunto(s)
Oligonucleótidos Fosforotioatos/química , Proteínas/química , Química Farmacéutica , Humanos , Oligonucleótidos Fosforotioatos/metabolismo , Proteínas/metabolismoRESUMEN
The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2'-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5'-terminal 2'-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein-protein interactions causative of cellular toxicity.
Asunto(s)
Ácidos Nucleicos/metabolismo , Oligonucleótidos Fosforotioatos/química , Proteínas/metabolismo , HumanosRESUMEN
Chemically modified antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages have been extensively studied as research and therapeutic agents. PS-ASOs can enter the cell and trigger cleavage of complementary RNA by RNase H1 even in the absence of transfection reagent. A number of cell surface proteins have been identified that bind PS-ASOs and mediate their cellular uptake; however, the mechanisms that lead to productive internalization of PS-ASOs are not well understood. Here, we characterized the interaction between PS-ASOs and epidermal growth factor receptor (EGFR). We found that PS-ASOs trafficked together with EGF and EGFR into clathrin-coated pit structures. Their co-localization was also observed at early endosomes and inside enlarged late endosomes. Reduction of EGFR decreased PS-ASO activity without affecting EGF-mediated signaling pathways and overexpression of EGFR increased PS-ASO activity in cells. Furthermore, reduction of EGFR delays PS-ASO trafficking from early to late endosomes. Thus, EGFR binds to PS-ASOs at the cell surface and mediates essential steps for active (productive) cellular uptake of PS-ASOs through its cargo-dependent trafficking processes which migrate PS-ASOs from early to late endosomes. This EGFR-mediated process can also serve as an additional model to better understand the mechanism of intracellular uptake and endosomal release of PS-ASOs.
Asunto(s)
Endocitosis/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos Fosforotioatos/genética , Transporte Biológico/genética , Membrana Celular/química , Membrana Celular/genética , Endosomas/química , Endosomas/genética , Factor de Crecimiento Epidérmico/genética , Receptores ErbB/química , Receptores ErbB/genética , Células HeLa , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Oligonucleótidos Antisentido/química , Oligonucleótidos Fosforotioatos/química , Unión Proteica , Transducción de Señal , TransfecciónRESUMEN
We reported previously that a 2' fluoro-modified (2' F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5-10-5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2'-O-(2-methoxyethyl) (2' MOE) or 2',4'-constrained 2'-O-ethyl (cEt) did. Rapid degradation of these proteins and cytotoxicity were observed in cells treated with 2' F PS-ASO. Here, we report that 2' F gapmer PS-ASOs of different sequences caused reduction in levels of DBHS proteins and hepatotoxicity in mice. 2' F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were observed as early as 24 to 48 hours after a single administration of the 2' F PS-ASO. The observed effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2' F nucleoside metabolites. Instead, we found that 2' F PS-ASO associated with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates positively with the 2' F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2' F PS-ASO in vivo.
Asunto(s)
Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Oligonucleótidos Antisentido/toxicidad , Oligonucleótidos Fosforotioatos/metabolismo , Transcriptoma/efectos de los fármacos , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Ratones Endogámicos BALB C , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Factor de Empalme Asociado a PTB/genética , Factor de Empalme Asociado a PTB/metabolismo , Oligonucleótidos Fosforotioatos/genética , Unión Proteica/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Transcriptoma/genéticaRESUMEN
RNase H1-dependent, phosphorothioate-modified antisense oligonucleotides (PS-ASOs) can enter cells through endocytic pathways and need to be released from the membrane-enclosed organelles, a limiting step for antisense activity. Accumulating evidence has suggested that productive PS-ASO release mainly occurs from late endosomes (LEs). However, how PS-ASOs escape from LEs is not well understood. Here, we report that upon PS-ASO incubation, COPII vesicles, normally involved in ER-Golgi transport, can re-locate to PS-ASO-containing LEs. Reduction of COPII coat proteins significantly decreased PS-ASO activity, without affecting the levels of PS-ASO uptake and early-to-late endosome transport, but caused slower PS-ASO release from LEs. COPII co-localization with PS-ASOs at LEs does not require de novo assembly of COPII at ER. Interestingly, reduction of STX5 and P115, proteins involved in tethering and fusion of COPII vesicles with Golgi membranes, impaired COPII re-localization to LEs and decreased PS-ASO activity. STX5 can re-locate to LEs upon PS-ASO incubation, can bind PS-ASOs, and the binding appears to be required for this pathway. Our study reveals a novel release pathway in which PS-ASO incubation causes LE re-localization of STX5, which mediates the recruitment of COPII vesicles to LEs to facilitate endosomal PS-ASO release, and identifies another key PS-ASO binding protein.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/fisiología , Endocitosis/fisiología , Endosomas/metabolismo , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/metabolismo , Vesículas Transportadoras/metabolismo , Células Cultivadas , Células HeLa , Células Hep G2 , Humanos , Transducción de SeñalRESUMEN
Antisense oligonucleotides (ASOs) are most commonly designed to reduce targeted RNA via RNase H1-dependent degradation, however kinetic parameters for ASO-mediated targeting and subsequent cleavage and degradation of RNA in living cells are poorly understood. In this manuscript we use an inducible minigene system to determine the time course of ASO activity in the cell. Estimates of the time required for the ASO to enter and traverse the cell, scan the target mRNA, bind the cognate site, recruit RNase H1 and initiate cleavage, are presented in the context of transcription and mRNA processing rates. Data are also presented which indicate that rates for RNase H1-dependent ASO-mediated degradation of the targeted RNAs are different for nuclear-retained versus RNAs exported to the cytoplasm and that the level of RNase H1 in the cell and cellular compartments is limiting to the rate of ASO activity. In both cellular compartments RNase H1 ASOs essentially double the endogenous rates of clearance of the target RNA. Overexpression of Escherichia coli RNase H1 or the presence of multiple cognate sites each further increase the rate of target RNA degradation.
Asunto(s)
Oligonucleótidos Antisentido/metabolismo , ARN Mensajero/metabolismo , Ribonucleasa H/metabolismo , Cinética , División del ARN , Procesamiento Postranscripcional del ARN , Transcripción GenéticaRESUMEN
While most siRNAs induce sequence-specific target mRNA cleavage and degradation in a process mediated by Ago2/RNA-induced silencing complex (RISC), certain siRNAs have also been demonstrated to direct target RNA reduction through deadenylation and subsequent degradation of target transcripts in a process which involves Ago1/RISC and P-bodies. In the current study, we present data suggesting that a third class of siRNA exist, which are capable of promoting target RNA reduction that is independent of both Ago and RISC. These siRNAs bind the target messenger RNA at the polyA signal and are capable of redirecting a small amount of polyadenylation to downstream polyA sites when present, however, the majority of the activity appears to be due to inhibition of polyadenylation or deadenylation of the transcript, followed by exosomal degradation of the immature mRNA.
Asunto(s)
Poliadenilación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Exorribonucleasas/metabolismo , Ratones , Proteínas de Unión al ARN/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , Receptores de Interleucina-4/genética , Receptores de Interleucina-4/metabolismoRESUMEN
U1 Adaptors are a recently reported novel approach for targeted reduction of mRNA transcripts. A U1 adaptor oligonucleotide comprising of a target-complimentary hybridization domain and a U1 recruitment domain, directs the U1 snRNP complex to the terminal exon of a targeted gene, subsequently inhibiting poly(A) tail addition and leading to degradation of that RNA species within the nucleus. Here, we present data demonstrating U1 adapter-mediated gene silencing can result in significant 'off-target' silencing effects as demonstrated by the reduction of multiple mRNA species that were not intended to be targeted. Our data suggest that a substantial portion of this U1 adaptor-mediated off-target mRNA reduction is the result of sequestration U1 snRNP at levels sufficient to affect splicing and processing of non-target transcripts.
Asunto(s)
Oligonucleótidos , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/antagonistas & inhibidores , Regulación hacia Abajo , Silenciador del Gen , Células HeLa , Humanos , Oligonucleótidos Antisentido , Procesamiento Postranscripcional del ARN , Empalme del ARN , ARN Nuclear Pequeño/antagonistas & inhibidoresRESUMEN
Hundreds of small nuclear non-coding RNAs, including small nucleolar RNAs (snoRNAs), have been identified in different organisms, with important implications in regulating gene expression and in human diseases. However, functionalizing these nuclear RNAs in mammalian cells remains challenging, due to methodological difficulties in depleting these RNAs, especially snoRNAs. Here we report a convenient and efficient approach to deplete snoRNA, small Cajal body RNA (scaRNA) and small nuclear RNA in human and mouse cells by conventional transfection of chemically modified antisense oligonucleotides (ASOs) that promote RNaseH-mediated cleavage of target RNAs. The levels of all seven tested snoRNA/scaRNAs and four snRNAs were reduced by 80-95%, accompanied by impaired endogenous functions of the target RNAs. ASO-targeting is highly specific, without affecting expression of the host genes where snoRNAs are embedded in the introns, nor affecting the levels of snoRNA isoforms with high sequence similarities. At least five snoRNAs could be depleted simultaneously. Importantly, snoRNAs could be dramatically depleted in mice by systematic administration of the ASOs. Together, our findings provide a convenient and efficient approach to characterize nuclear non-coding RNAs in mammalian cells, and to develop antisense drugs against disease-causing non-coding RNAs.
Asunto(s)
Técnicas de Silenciamiento del Gen , Oligonucleótidos Antisentido , ARN Nuclear Pequeño/metabolismo , Animales , Secuencia de Bases , Línea Celular , Núcleo Celular/química , Células HeLa , Humanos , Ratones , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/administración & dosificación , Oligonucleótidos Antisentido/análisis , Oligonucleótidos Antisentido/química , ARN Ribosómico/metabolismo , ARN Nucleolar Pequeño/antagonistas & inhibidores , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/metabolismo , TransfecciónRESUMEN
RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.
Asunto(s)
Oligonucleótidos Antisentido , Oligonucleótidos Fosforotioatos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Fosforotioatos/genética , Oligonucleótidos Fosforotioatos/metabolismo , Oligonucleótidos Fosforotioatos/farmacología , Precursores del ARN , Ribonucleasa H/genética , Ribonucleasa H/metabolismoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is caused by point mutations that increase utilization of an alternate splice donor site in exon 11 of LMNA (the gene encoding lamin C and prelamin A). The alternate splicing reduces transcripts for wild-type prelamin A and increases transcripts for a truncated prelamin A (progerin). Here, we show that antisense oligonucleotides (ASOs) against exon 11 sequences downstream from the exon 11 splice donor site promote alternate splicing in both wild-type and HGPS fibroblasts, increasing the synthesis of progerin. Indeed, wild-type fibroblasts transfected with these ASOs exhibit progerin levels similar to (or greater than) those in fibroblasts from HGPS patients. This progerin was farnesylated, as judged by metabolic labeling studies. The synthesis of progerin in wild-type fibroblasts was accompanied by the same nuclear shape and gene-expression perturbations observed in HGPS fibroblasts. An ASO corresponding to the 5' portion of intron 11 also promoted alternate splicing. In contrast, an ASO against exon 11 sequences 5' to the alternate splice site reduced alternate splicing in HGPS cells and modestly lowered progerin levels. Thus, different ASOs can be used to increase or decrease 'HGPS splicing'. ASOs represent a new and powerful tool for recreating HGPS pathophysiology in wild-type cells.
Asunto(s)
Mutación , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Oligonucleótidos Antisentido/uso terapéutico , Progeria/genética , Progeria/terapia , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/genética , Empalme Alternativo , Secuencia de Bases , Línea Celular , Células Cultivadas , Exones , Fibroblastos/metabolismo , Terapia Genética , Humanos , Lamina Tipo A , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Progeria/metabolismoRESUMEN
Survival of motor neuron 2, centromeric (SMN2) is a gene that modifies the severity of spinal muscular atrophy (SMA), a motor-neuron disease that is the leading genetic cause of infant mortality. Increasing inclusion of SMN2 exon 7, which is predominantly skipped, holds promise to treat or possibly cure SMA; one practical strategy is the disruption of splicing silencers that impair exon 7 recognition. By using an antisense oligonucleotide (ASO)-tiling method, we systematically screened the proximal intronic regions flanking exon 7 and identified two intronic splicing silencers (ISSs): one in intron 6 and a recently described one in intron 7. We analyzed the intron 7 ISS by mutagenesis, coupled with splicing assays, RNA-affinity chromatography, and protein overexpression, and found two tandem hnRNP A1/A2 motifs within the ISS that are responsible for its inhibitory character. Mutations in these two motifs, or ASOs that block them, promote very efficient exon 7 inclusion. We screened 31 ASOs in this region and selected two optimal ones to test in human SMN2 transgenic mice. Both ASOs strongly increased hSMN2 exon 7 inclusion in the liver and kidney of the transgenic animals. Our results show that the high-resolution ASO-tiling approach can identify cis-elements that modulate splicing positively or negatively. Most importantly, our results highlight the therapeutic potential of some of these ASOs in the context of SMA.