RESUMO
Translation initiation of the hepatitis C virus (HCV) mRNA depends on an internal ribosome entry site (IRES) that encompasses most of the 5'UTR and includes nucleotides of the core coding region. This study shows that the polypyrimidine-tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the HCV 5'UTR, stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Our results show that PTB1 and PTB4, but not PTB2, stimulate HCV IRES activity in HuH-7 and HEK293T cells. In HuH-7 cells, PTB1 promotes HCV IRES-mediated initiation more strongly than PTB4. Mutations in PTB1, PTB4, RRM1/RRM2, or RRM3/RRM4, which disrupt the RRM's ability to bind RNA, abrogated the protein's capacity to stimulate HCV IRES activity in HuH-7 cells. In HEK293T cells, PTB1 and PTB4 stimulate HCV IRES activity to similar levels. In HEK293T cells, mutations in RRM1/RRM2 did not impact PTB1's ability to promote HCV IRES activity; and mutations in PTB1 RRM3/RRM4 domains reduced, but did not abolish, the protein's capacity to stimulate HCV IRES activity. In HEK293T cells, mutations in PTB4 RRM1/RRM2 abrogated the protein's ability to promote HCV IRES activity, and mutations in RRM3/RRM4 have no impact on PTB4 ability to enhance HCV IRES activity. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity in a cell type-specific manner. We conclude that PTB1 and PTB4, but not PTB2, act as IRES transacting factors of the HCV IRES.
Assuntos
Hepatite C , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Humanos , Regiões 5' não Traduzidas , Células HEK293 , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/genética , Sítios Internos de Entrada Ribossomal , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
The 5' untranslated region (UTR) of the full-length mRNA of the mouse mammary tumor virus (MMTV) harbors an internal ribosomal entry site (IRES). In this study, we show that the polypyrimidine tract-binding protein (PTB), an RNA-binding protein with four RNA recognition motifs (RRMs), binds to the MMTV 5' UTR stimulating its IRES activity. There are three isoforms of PTB: PTB1, PTB2, and PTB4. Results show that PTB1 and PTB4, but not PTB2, stimulate MMTV-IRES activity. PTB1 promotes MMTV-IRES-mediated initiation more strongly than PTB4. When expressed in combination, PTB1 further enhanced PTB4 stimulation of the MMTV-IRES, while PTB2 fully abrogates PTB4-induced stimulation. PTB1-induced stimulation of MMTV-IRES was not altered in the presence of PTB4 or PTB2. Mutational analysis reveals that stimulation of MMTV-IRES activity is abrogated when PTB1 is mutated either in RRM1/RRM2 or RRM3/RRM4. In contrast, a PTB4 RRM1/RRM2 mutant has reduced effect over MMTV-IRES activity, while stimulation of the MMTV-IRES activity is still observed when the PTB4 RRM3/RMM4 mutant is used. Therefore, PTB1 and PTB4 differentially stimulate the IRES activity. In contrast, PTB2 acts as a negative modulator of PTB4-induced stimulation of MMTV-IRES. We conclude that PTB1 and PTB4 act as IRES trans-acting factors of the MMTV-IRES.
Assuntos
Regiões 5' não Traduzidas , Vírus do Tumor Mamário do Camundongo/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Capuzes de RNA , RNA Mensageiro/genética , Sítios de Ligação , Técnicas de Silenciamento de Genes , Genes Virais , Células HEK293 , Humanos , Sítios Internos de Entrada Ribossomal , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genéticaRESUMO
Trypanosomes regulate gene expression mostly by posttranscriptional mechanisms, including control of mRNA turnover and translation efficiency. This regulation is carried out via certain elements located at the 3'-untranslated regions of mRNAs, which are recognized by RNA-binding proteins. In trypanosomes, trans-splicing is of central importance to control mRNA maturation. We have previously shown that TcDRBD4/PTB2, a trypanosome homolog of the human polypyrimidine tract-binding protein splicing regulator, interacts with the intergenic region of one specific dicistronic transcript, referred to as TcUBP (and encoding for TcUBP1 and TcUBP2, two closely kinetoplastid-specific proteins). In this work, a survey of TcUBP RNA processing revealed certain TcDRBD4/PTB2-regulatory elements within its intercistronic region, which are likely to influence the trans-splicing rate of monocistronic-derived transcripts. Furthermore, TcDRBD4/PTB2 overexpression in epimastigote cells notably decreased both UBP1 and UBP2 protein expression. This type of posttranscriptional gene regulatory mechanism could be extended to other transcripts as well, as we identified several other RNA precursor molecules that specifically bind to TcDRBD4/PTB2. Altogether, these findings support a model in which TcDRBD4/PTB2-containing ribonucleoprotein complexes can prevent trans-splicing. This could represent another stage of gene expression regulation mediated by the masking of trans-splicing/polyadenylation signals.
Assuntos
Regulação da Expressão Gênica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas de Protozoários/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Animais , DNA Intergênico/genética , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas de Protozoários/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/genéticaRESUMO
Objective: To present a detailed explanation on the processing of magnetic susceptibility weighted imaging (SWI), demonstrating the effects of echo time and sensitive mask on the differentiation between calcification and hemosiderin. Materials and Methods: Computed tomography and magnetic resonance (magnitude and phase) images of six patients (age range 41– 54 years; four men) were retrospectively selected. The SWI images processing was performed using the Matlab’s own routine. Results: Four out of the six patients showed calcifications at computed tomography images and their SWI images demonstrated hyperintense signal at the calcification regions. The other patients did not show any calcifications at computed tomography, and SWI revealed the presence of hemosiderin deposits with hypointense signal. Conclusion: The selection of echo time and of the mask may change all the information on SWI images, and compromise the diagnostic reliability. Amongst the possible masks, the authors highlight that the sigmoid mask allows for contrasting calcifications and hemosiderin on a single SWI image. .
Objetivo: Expor em detalhes o processamento da imagem ponderada em suscetibilidade magnética (susceptibility weighted imaging – SWI), destacando o efeito da escolha do tempo de eco e da máscara sensível à diferenciação de calcificação e hemossiderina simultaneamente. Materiais e Métodos: Imagens de tomografia computadorizada e por ressonância magnética (magnitude e fase) foram selecionadas, retrospectivamente, de seis pacientes (idades entre 41 e 54 anos; quatro homens). O processamento das imagens SWI foi realizado em rotina própria no programa Matlab. Resultados: Dos seis pacientes estudados, quatro apresentaram calcificações nas imagens de tomografia computadorizada. Nestes, as imagens SWI mostraram sinal hiperintenso para as regiões de calcificações. Os outros dois pacientes não apresentaram calcificações nas imagens de tomografia computadorizada e apresentaram depósito de hemossiderina com sinal hipointenso na imagem SWI. Conclusão: A escolha do tempo de eco e da máscara pode alterar toda a informação da imagem SWI e comprometer a confiabilidade diagnóstica. Dentre as possíveis máscaras, destacamos que a máscara sigmoide permite contrastar calcificação e hemossiderina em uma única imagem SWI. .
Assuntos
Animais , Camundongos , Processamento Alternativo/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Tropomiosina/genética , Sequência de Bases , Sítios de Ligação , Primers do DNA , Éxons , Vetores Genéticos , Ligantes , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/metabolismo , TransfecçãoRESUMO
The 3' untranslated region (3'UTR) of the dengue virus (DENV) genome contain several sequences required for translation, replication and cyclization processes. This region also binds cellular proteins such as La, polypyrimidine tract-binding protein (PTB), Y box-binding protein 1, poly(A)-binding protein and the translation initiation factor eEF-1 alpha. PTB is a cellular protein that interacts with the regulatory sequences of positive-strand RNA viruses such as several picornaviruses and hepatitis C virus. In the present report, it was demonstrated that PTB translocates from the nucleus to the cytoplasm during DENV infection. At 48 h post-infection, PTB, as well as the DENV proteins NS1 and NS3, were found to co-localize with the endoplasmic reticulum marker calnexin. Silencing of PTB expression inhibited virus translation and replication, whilst overexpression of PTB augmented these processes. Thus, these results provide evidence that, during infection, PTB moves from the nucleus to the cytoplasm and plays an important role in the DENV replicative cycle.