Abstract 3974: Nucleolin interacts with the microprocessor complex to affect the biogenesis of miR-15a and miR-16-1

Abstract

Abstract Purpose: To investigate the role of nucleolin in microRNA regulation. Experimental Procedure: Nucleolin was overexpressed and knocked down followed by miRNA expression analysis by qRT-PCR. Nucleolin cellular localization was altered by treatment with parthenolide and verified by cellular fractionation. A change in primary, precursor, and mature microRNA following altered nucleolin localization was determined by endpoint PCR, northern blot, and qRT-PCR, respectively. Immunoprecipitation was utilized to investigate the interaction of nucleolin with the components of the microprocessor complex, Drosha and DGCR8. To determine the interaction of nucleolin with primary microRNA, RNA immunoprecipitation was performed. Results: Altered expression of nucleolin positively correlated with the expression of mature miR-15a and miR-16-1. Upon induction of cytoplasmic nucleolin, mature and precursor miR-15/16 species significantly decreased, while primary species increased. When nuclear levels of nucleolin increased following treatment with parthenolide, miR-15/16 dramatically and significantly increased. However, after nucleolin was knocked down, treatment with parthenolide failed to increase mature miR-15/16. Additionally, primary species were depleted in the presence of nucleolin, indicating increased processing; yet in the absence of nucleolin primary species persisted, indicating a failure to process primary miRNA. Immunoprecipitation studies revealed that nucleolin interacts with both Drosha and DGCR8. This interaction is independent of RNA as it persisted following RNase treatment. RNA immunoprecipitation revealed that nucleolin bound to primary miR-15/16 to a level equal to DGCR8. Conclusions: Nucleolin expression levels do indeed alter miR-15/16 levels. In addition to absolute levels of nucleolin, cellular localization is also important. We conclude that cytoplasmic nucleolin increased primary miRNA while decreasing precursor and mature miRNA due to spatial segregation from the microprocessor complex involved in primary miRNA processing. Increased nuclear nucleolin greatly increased mature miR-15/16 likely by facilitating nucleolin's interaction with the microprocessor complex. Immunoprecipitation data revealed that nucleolin interacts with Drosha and DGCR8 and this interaction is not due to tethering to RNA as it persisted despite RNase treatment. Lastly, nucleolin, which is an RNA binding protein, can bind to primary miRNA. Nucleolin may facilitate processing of primary miRNA by positioning it for cleavage by Drosha, as is nucleolin's role in the biogenesis of ribosomal RNA. Nucleolin is known to localize to the cytoplasm of numerous cancers including CLL and breast cancer. Our data indicates this may lead to a decrease of the tumor-suppressor miR-15/16 providing a survival advantage for cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3974. doi:10.1158/1538-7445.AM2011-3974