SUM Oylation Of DGCR8 And Its Function In MiRNA Pathway

DGCR8, the major partner of Drosha, plays an important role in miRNA biogenesis generally via interacting with pri-miRNA and recruiting Drosha to cleave pri-miRNA at the correct site into pre-mi RNA. Then the pre-miRNA is cleaved by Dicer to mature miRNA duplex. Deletion or abnormal expression of DGCR8 may affect the processing activity of Drosha, which would decrease the amount of mature mi RNAs and lead to the emergence of disease. Recently it has been reported that deacetylation of DGCR8 dsRBDs by HDAC1 enhances its affinity with pri-miRNAs. While the protein level of DGCR8 is upregulated by its phosphorylation and is downregulated by Drosha via cutting the mRNA of DGCR8. Though the molecular mechanisms of DGCR8 in miRNA biogenesis is relatively known, other functions and regulations of DGCR8 remain to be explored.Mature miRNA has been widely accepted as the only executor in miRNA pathway. But interestingly, Chen CZ’s group and Kay MA’s group have discovered that both pri- and pre-miRNA can recognize and repress target genes. This provides a prospect of brand-new functioning pathways in miRNA regulation. In this study we found that DGCR8 was modified by SUMO1, and this modification enhanced DGCR8’s stability and affinity with pri-mi RNA together with an increased target repression mediated directly by pri-miRNAs.In the first part of our study, we proved that DGCR8 was modified by SUMO1 and SUMOylation of DGCR8 was enhanced by its phosphorylation. Mutation at K707, which was identified as the major SUMOylation site of DGCR8, downregulated the protein level by increasing its ubiquitination. We also proved that the interaction between DGCR8-K707 R and pri-miRNA was decreased. Though there was no change in miRNA biogenesis with DGCR8-K707 R mutation, a weakened gene silencing effect induced directly by pri-miRNA was observed and the decreased colony formation, cell migration or tumor growth were exhibited in PC3 or A549 cells stably expressing DGCR8-K707 R.In the second part of our study, we found that SUMO1 modification of DGCR8 was promoted by p14 ARF via protein interaction, and K259 which was within the association region between DGCR8 and p14 ARF was identified as another major SUMO1 modification site. Similar with DGCR8-K707 R, mutation at K259 decreased the protein stability, affinity with pri-miRNA and weakened gene silencing effect. However, in contrast with DGCR8-K707 R, a localization change of DGCR8-K259 R from nuclear to cytoplasm was obtained, and an increased colony formation, cell migration, tumor growth were displayed in A549 cells when DGCR8-K259 R was stably expressing.