RESUMO
In recent years, non-coding RNAs (ncRNAs) have become an exciting area of research. It has been demonstrated that ncRNAs play an important role in the regulation of gene expression in eukaryotic cells. However, little is known about ncRNAs in lymphocytes. In this study, we investigated the presence of ncRNAs in lymphocytes of C57BL/6 mice bearing B16 melanoma by using the differential display reverse transcription-PCR (DD-RT-PCR). PKR is a serine/threonine kinase containing two RNA-binding domains within the N-terminal region. We took advantage of the ability of RNAs to bind PKR in order to identify ncRNAs of lymphocytes activated during tumor development. Thus, RNAs that co-immunoprecipitated with PKR were reversed transcribed, re-amplified, cloned, sequenced and the secondary structure was determined. The ability of transcripts obtained by in vitro transcription to activate PKR was also examined. We detected a highly structured transcript of 220 nt with no open reading frame (ORF) which is able to activate PKR, and it is only expressed in lymphocytes of C57BL/6 mice bearing B16 melanoma. Therefore, the 220 nt transcript may be included in the class of ncRNAs that act by modifying protein activity and our data suggest that regulation of gene expression in activated lymphocytes by this ncRNA could be mediated by PKR through the activation of the transcription factor NF-kappaB.
Assuntos
Linfócitos/metabolismo , Melanoma Experimental/metabolismo , Neoplasias/metabolismo , RNA não Traduzido/genética , eIF-2 Quinase/metabolismo , Animais , Sequência de Bases , Linhagem Celular Tumoral , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA não Traduzido/metabolismo , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
It has been known for decades that exogenous RNAs are able to induce cytotoxic T lymphocytes (CTLs) and immunological reactivity to a wide variety of antigens. The molecular events responsible for these effects remain unclear for more than two decades. It has been decided to revisit this phenomenon in the light of new concepts that are just emerging in Molecular Biology, such as the regulation of gene expression by noncoding RNAs, named regulatory RNAs. The immunological effects observed in lymphocytes treated with RNAs obtained from lymph nodes of immunized animals with different types of antigens including synthetic peptides of the human immunodeficiency virus type 1 (HIV-1) have been investigated. Our recent results showed that regulatory RNAs are involved in this phenomenon, which is due to the activation of the RNA-dependent protein kinase (PKR) by regulatory RNAs with subsequent activation of the transcription factor NF-kappaB. The RNA-dependent protein kinase (PKR) is a serine/threonine kinase and contains two RNA-binding domains (RBD-I and RBD-II) within the N-terminal region. PKR is activated by viral double-stranded RNA (dsRNA) and highly structured single-stranded RNAs. This review will focus on the structure and functions of PKR including its role in HIV-1 infection. Special emphasis will be placed on a regulatory RNA, named p9-RNA, isolated from lymphocytes of animals immunized with the synthetic peptide p9 (pol: 476-484) of HIV-1. It was found that the regulatory p9-RNA induces CTLs and production of IFN-gamma. These findings showed for the first time that transcriptional control of gene expression by a regulatory RNA can be mediated by PKR through the activation of the transcription factor NF-kappaB. A model for the mechanism of action of the regulatory p9-RNA responsible for the production of IFN-gamma is proposed. Elucidating the molecular mechanism of p9-RNA may contribute to determining the rationale for the use of this regulatory RNA as an immunomodulator in HIV-infected patients.
Assuntos
Infecções por HIV/imunologia , Linfócitos/enzimologia , Linfócitos/imunologia , RNA não Traduzido/fisiologia , eIF-2 Quinase/metabolismo , Animais , Humanos , Linfócitos/metabolismo , RNA , eIF-2 Quinase/químicaRESUMO
RNA-dependent protein kinase (PKR) mediates the antiviral activity of interferon and also has implications in cell growth, differentiation, and apoptosis. On the other hand, the tumor suppressor function of PKR is still controversial. PKR is a serine/threonine kinase that contains two RNA-binding domains (RBD-I and RBD-II) and RBD-I is critical for its activation. Site-directed mutagenesis studies indicated that a single amino acid substitution in RBD-I is sufficient to abolish the interaction of human PKR with RNA. Also, PKR mutants that are unable to bind RNA are inactive in vitro and have no antiproliferative activity in vivo. There have been no reports of mutations in the RNA-binding domains of PKR of tumor cells taken directly from patients. We investigated the presence of mutations in the RBD-I and RBD-II of PKR gene in children with acute lymphoblastic leukemia (ALL). The RNA extracted from bone marrow samples of 15 patients with ALL (5 patients T-lineage; 10 patients B-lineage) was used for to synthesize cDNA and amplify the sequences corresponding to RBD-I and RBD-II. The PCR products were subsequently cloned and sequenced. A point mutation was detected in the RBD-I of PKR from a patient with ALL of T-cell lineage that is located at cDNA nt 50 A --> G (17 Tyr-->Cys). We also found that activation of a PKR mutant by the polyinosinic acid:polycytidylic acid (poly I:C) is impaired when compared with the wild-type PKR. Additional work is required to elucidate whether this point mutation plays a role in the formation and/or maintenance of leukemic cells. To our knowledge, this study is the first example of detection of a mutation in the RBD-I of PKR gene from tumor cells taken directly from patients.
Assuntos
Mutação Puntual/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA/metabolismo , eIF-2 Quinase/química , eIF-2 Quinase/metabolismo , Sequência de Bases , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Poli I-C/farmacologia , eIF-2 Quinase/genéticaRESUMO
Previous results with p9-RNA, obtained from lymph nodes of animals immunized with the peptide p9 of HIV-1, suggested that its effects on lymphocytes could be mediated by RNA-dependent protein kinase (PKR). Here we report that p9-RNA activates PKR leading to the degradation of the inhibitor I-kappaB alpha and the concomitant nuclear factor kappa B (NF-kappaB) activation. The fractionation of p9-RNA by affinity chromatography indicates that the poly A(+) p9-RNA is the fraction responsible for PKR activation. We also found that p9-RNA induces the production of interferon-gamma (IFN-gamma), but not interleukin (IL-4) since only IFN-gamma gene promoter contains NF-kappaB binding site. This study provides the first evidence that transcriptional control of gene expression by regulatory RNAs can be mediated by PKR through NF-kappaB activation. A model for the mechanism of action of poly A(+) p9-RNA is proposed.