RESUMEN
3' UTRs play important roles in the gene regulation network via their influence on mRNA stability, translational efficiency, and subcellular localization. For a given gene, 3' UTRs of different lengths generated by alternative polyadenylation (APA) may result in functional differences in regulation. The mechanistic details of how length changes of 3' UTRs alter gene function remain unclear. By combining APA sequencing and polysome profiling, we observed that mRNA isoforms with shorter 3' UTRs were bound with more polysomes in six cell lines but not in NIH3T3 cells, suggesting that changing 3' UTRs to shorter isoforms may lead to a higher gene translational efficiency. By interfering with the expression of TNRC6A and analyzing AGO2-PAR-CLIP data, we revealed that the APA effect on translational efficiency was mainly regulated by miRNAs, and this regulation was cell cycle dependent. The discrepancy between NIH3T3 and other cell lines was due to contact inhibition of NIH3T3. Thus, the crosstalk between APA and miRNAs may be needed for the regulation of protein translational efficiency.
Asunto(s)
MicroARNs/genética , Poliadenilación , Biosíntesis de Proteínas , Regiones no Traducidas 3' , Células 3T3 , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Ciclo Celular , Células Cultivadas , Humanos , Células MCF-7 , Ratones , Polirribosomas/metabolismo , Señales de Poliadenilación de ARN 3' , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Especificidad de la EspecieRESUMEN
Global shortening of 3'UTRs by alternative polyadenylation (APA) has been observed in cancer cells. However, the role of APA in cancer remains unknown. CCND1 is a proto-oncogene that regulates progression through the G1-S phase of the cell cycle; moreover, it has been observed to be switching to proximal APA sites in cancer cells. To investigate the biological function of the APA of CCND1, we edited the weak poly(A) signal (PAS) of the proximal APA site to a canonical PAS using the CRISPR/Cas9 method, which can force the cells to use a proximal APA site. Cell cycle profiling and proliferation assays revealed that the proximal APA sites of CCND1 accelerated the cell cycle and promoted cell proliferation, but UTR-APA and CR-APA act via different molecular mechanisms. These results indicate that PAS editing with CRISPR/Cas9 provides a good method by which to study the biological function of APA.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , Ciclina D1/metabolismo , Poliadenilación/fisiología , Regiones no Traducidas 3'/fisiología , Sistemas CRISPR-Cas/fisiología , Proliferación Celular/fisiología , Ciclina D1/genética , Sitios Genéticos , Vectores Genéticos , Células HEK293 , Humanos , Mutación , Sistemas de Lectura Abierta/fisiología , Poli A/metabolismo , Isoformas de Proteínas/metabolismo , Proto-Oncogenes Mas , ARN Mensajero/metabolismo , Regiones no Traducidas/fisiologíaRESUMEN
This study aimed to assess the effect of toluidine blue (TB)-mediated photodynamic inactivation of periodontal pathogens (PP) from periodontopathic patients. Photodynamic therapy (PDT) was carried out using TB and 635 nm laser light irradiation. The bactericidal effect was evaluated, and important PDT parameters including light intensity, energy dose, and TB concentration were determined. Our findings suggest that TB-mediated lethal photosensitization of PP in vivo is possible. However, to obtain ideal bactericidal effect, higher doses of light and photosensitizer should be required in treatment in vivo than their planktonic counterparts. The best therapeutic effect was observed in treatment by 1 mg/ml TB combined with 12 J/cm(2) at 159 mW/cm(2) light irradiation. Moreover, because of the considerable interindividual differences of bacterial populations, TB-mediated PDT might not be equally effective among periodontopathic patients, and further studies on improvement of this therapeutic modality is needed.