Structure and Function of the Exon Junction Complex in Arabidopsis thaliana

Puerto Madryn

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2010

The exon junction complex (EJC) is deposited onto mRNAs during splicing and directs post-transcriptional processes in the cytoplasm. In this work, we studied three EJC components in Arabidopsis thaliana: AtMago, AtY14 and AtPym. Plants that contain the b- glucuronidase (gus) gene under the control of the respective promoter sequences show expression mainly in meristematic regions. A bioinformatic search allowed the identification of site II motifs (TGGGCC/T) that profoundly affected the expression of the genes when mutated and were recognized by proteins present in nuclear extracts. In addition, over-expression of AtMago or AtY14 in plants produced an increase in the amount of AtPym, as indicated by western blots. In order to determine if these proteins were modified post-translationally, we carried out both in vitro and in vivo phosphorylation assays and we detected that AtY14 and AtPym could be phosphorylated by proteins present in plant extracts. Pulldown assays showed that this modification plays an important role in regulating complex formation between these proteins. Transient transformation assays indicated that the expression of each of the proteins under study produces an increase in the expression of genes that require an intron for maximal expression, suggesting that the exon junction complex participates in intron-mediated enhancement of expression in plants.Arabidopsis thaliana: AtMago, AtY14 and AtPym. Plants that contain the b- glucuronidase (gus) gene under the control of the respective promoter sequences show expression mainly in meristematic regions. A bioinformatic search allowed the identification of site II motifs (TGGGCC/T) that profoundly affected the expression of the genes when mutated and were recognized by proteins present in nuclear extracts. In addition, over-expression of AtMago or AtY14 in plants produced an increase in the amount of AtPym, as indicated by western blots. In order to determine if these proteins were modified post-translationally, we carried out both in vitro and in vivo phosphorylation assays and we detected that AtY14 and AtPym could be phosphorylated by proteins present in plant extracts. Pulldown assays showed that this modification plays an important role in regulating complex formation between these proteins. Transient transformation assays indicated that the expression of each of the proteins under study produces an increase in the expression of genes that require an intron for maximal expression, suggesting that the exon junction complex participates in intron-mediated enhancement of expression in plants.: AtMago, AtY14 and AtPym. Plants that contain the b- glucuronidase (gus) gene under the control of the respective promoter sequences show expression mainly in meristematic regions. A bioinformatic search allowed the identification of site II motifs (TGGGCC/T) that profoundly affected the expression of the genes when mutated and were recognized by proteins present in nuclear extracts. In addition, over-expression of AtMago or AtY14 in plants produced an increase in the amount of AtPym, as indicated by western blots. In order to determine if these proteins were modified post-translationally, we carried out both in vitro and in vivo phosphorylation assays and we detected that AtY14 and AtPym could be phosphorylated by proteins present in plant extracts. Pulldown assays showed that this modification plays an important role in regulating complex formation between these proteins. Transient transformation assays indicated that the expression of each of the proteins under study produces an increase in the expression of genes that require an intron for maximal expression, suggesting that the exon junction complex participates in intron-mediated enhancement of expression in plants.