RESUMO
Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and ß-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells.
Assuntos
Poli A/genética , Poliadenilação , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Sequência de Aminoácidos , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Células HEK293 , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Humanos , Lipídeos/química , Dados de Sequência Molecular , Plasmídeos/química , Plasmídeos/metabolismo , Poli A/metabolismo , Polinucleotídeo Adenililtransferase , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Elementos de Resposta , Transdução de Sinais , Transfecção , beta Catenina/genética , beta Catenina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
ABSTRACT: Hospital-acquired infections caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli are a global problem. Healthy people can carry ESBL-producing E. coli in the intestines; thus, E. coli from healthy people can potentially cause hospital-acquired infections. Therefore, the transmission routes of ESBL-producing E. coli from healthy persons should be determined. A foodborne outbreak of human norovirus (HuNoV) GII occurred at a restaurant in Shizuoka, Japan, in 2018. E. coli O25:H4 was isolated from some of the HuNoV-infected customers. Pulsed-field gel electrophoresis showed that these E. coli O25:H4 strains originated from one clone. Because the only epidemiological link among the customers was eating food from this restaurant, the customers were concurrently infected with E. coli O25:H4 and HuNoV GII via the restaurant food. Whole genome analysis revealed that the E. coli O25:H4 strains possessed genes for regulating intracellular iron and expressing the flagellum and flagella. Extraintestinal pathogenic E. coli often express these genes on the chromosome. Additionally, the E. coli O25:H4 strains had plasmids harboring nine antimicrobial resistance genes. These strains harbored ESBL-encoding blaCTX-M-14 genes on two loci of the chromosome and had higher ESBL activity. Multilocus sequence typing and fimH subtyping revealed that the E. coli O25:H4 strains from the outbreak belonged to the subclonal group, ST131-fimH30R, which has been driving ESBL epidemics in Japan. Because the E. coli O25:H4 strains isolated in the outbreak belonged to a subclonal group spreading in Japan, foods contaminated with ESBL-producing E. coli might contribute to spreading these strains among healthy persons. The isolated E. coli O25:H4 strains produced ESBL and contained plasmids with multiple antimicrobial resistance genes, which may make it difficult to select antimicrobials for treating extraintestinal infections caused by these strains.