RESUMO
Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/genética , Oligonucleotídeos/farmacologia , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Processamento Alternativo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Glioblastoma/genética , Glioblastoma/patologia , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/genética , Músculo Esquelético/crescimento & desenvolvimento , Degradação do RNAm Mediada por Códon sem Sentido , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/antagonistas & inibidores , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de Serina-Arginina/antagonistas & inibidores , Fatores de Processamento de Serina-Arginina/metabolismo , Sequências de Repetição em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
The gene encoding the kinase Mnk2 (MKNK2) is alternatively spliced to produce two isoforms-Mnk2a and Mnk2b. We previously showed that Mnk2a is downregulated in several types of cancer and acts as a tumor suppressor by activation of the p38-MAPK stress pathway, inducing apoptosis. Moreover, Mnk2a overexpression suppressed Ras-induced transformation in culture and in vivo. In contrast, the Mnk2b isoform acts as a pro-oncogenic factor. In this study, we designed modified-RNA antisense oligonucleotides and screened for those that specifically induce a strong switch in alternative splicing of the MKNK2 gene (splice switching oligonucleotides or SSOs), elevating the tumor suppressive isoform Mnk2a at the expense of the pro-oncogenic isoform Mnk2b. Induction of Mnk2a by SSOs in glioblastoma cells activated the p38-MAPK pathway, inhibited the oncogenic properties of the cells, re-sensitized the cells to chemotherapy and inhibited glioblastoma development in vivo. Moreover, inhibition of p38-MAPK partially rescued glioblastoma cells suggesting that most of the anti-oncogenic activity of the SSO is mediated by activation of this pathway. These results suggest that manipulation of MKNK2 alternative splicing by SSOs is a novel approach to inhibit glioblastoma tumorigenesis.
Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oligonucleotídeos/genética , Proteínas Serina-Treonina Quinases/genética , Processamento Alternativo , Animais , Apoptose , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Genes Supressores de Tumor , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Oligonucleotídeos Antissenso , Fosforilação , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Intrinsically disordered regions (IDRs) in proteins are highly abundant, but they are still commonly viewed as long stretches of polar, solvent-accessible residues. Here we show that the disordered C-terminal domain (CTD) of HIV-1 Rev has two subregions that carry out two distinct complementary roles of regulating protein oligomerization and contributing to stability. We propose that this takes place through a delicate balance between charged and hydrophobic residues within the IDR. This means that mutations in this region, as well as the known mutations in the structured region of the protein, can affect protein function. We suggest that IDRs in proteins should be divided into subdomains similarly to structured regions, rather than being viewed as long flexible stretches.
Assuntos
HIV-1/química , Proteínas Intrinsicamente Desordenadas/química , Produtos do Gene rev do Vírus da Imunodeficiência Humana/química , Infecções por HIV/virologia , HIV-1/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Estabilidade Proteica , Produtos do Gene rev do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain plant-type ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (â¼20 Å), it leads to a significant change in the redox potential of the iron-sulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.
