Nucleolin's Role in the Biogenesis of Pri-miRNA 16 by the Microprocessor Complex: An In silico Approach.

MicroRNAs (miRNAs) are short, non-coding strands of RNA found in eukaryotes. They play a role in gene silencing through inhibiting translation or degrading mRNA. Primary miRNA (pri-miRNA) processing is the initial step in producing a mature microRNA molecule. The microprocessor complex (MP) proteins, DROSHA and DGCR8, cleaves the pri-miRNA molecule to a shorter hairpin structure. This cleaved RNA hairpin, precursor miRNA, is sent to the cytoplasm for further processing into the mature miRNA. Previous research has uncovered that the expression and cellular localization of the stress-responsive protein, nucleolin (NCL) affects the processing of several miRNAs, including microRNA 16 (miR16). Nucleolin is an RNA binding protein and is primarily located in the nucleolus but can also be localized in various cellular compartments. When NCL is present in the nucleus, the MP complex works more efficiently in cleaving pri-miR 16-1 to its pre-miRNA form resulting in an upregulation of miR 16-1. It has been proposed that nucleolin binds to pri-miR 16-1 and stabilizes its confirmation for cleavage by the MP complex. However, the mechanistic and structural details on how nucleolin interacts with pri-miRNA 16-1 or the MP are still unclear. It is also unknown if NCL is similarly involved in miR 16-2 biogenesis. NCL and miRNA 16 are also intricately involved in the regulation of the expression of bcl2, an anti-apoptotic and oncogenic protein. When NCL is in the cytoplasm, it stabilizes the 3' untranslated region of bcl2 mRNA leading to an increased expression of the protein. Unlike nucleolin, miR 16 represses the translation of bcl2 mRNA. At high levels of miR 16 when NCL is localized to the nucleus, bcl2 protein expression is low. When cellular stress occurs, NCL returns to the cytoplasm to stabilize bcl2 and this also results in less efficiency in pri-miR 16 processing, resulting in an overall increase in bcl2 protein. It is evident that NCL balances the levels of bcl2 and miR16 through its cellular localization. Since the molecular mechanism underlying NCL's role in the MP-based processing of pri-miR16-1 remains largely unknown, in this study, we used computational approaches including structural alignments, protein-protein, and protein-RNA docking analyses to determine how pri-miR 16-1, nucleolin, and the MP complex interact. In addition, we have investigated if pri-miR 16-2 interacts with the NCL and the MP complex in a similar fashion. We show in our predictive models that the interaction interface of pri-miR 16-1 and pri-miR 16-2 with nucleolin and the MP complex nucleolin is distinct. For pri-miR 16-1, the interaction interface involves NCL and DGCR8 B and C, but in the case of pri-miR 16-2, it involves NCL and DROSHA. Additionally, in the presence of nucleolin, the hydrogen bond interactions are reduced between pri-miR 16-2 and the MP complex. Although the number of interactions between the different components of the MP complex is the same in both scenarios, the specific residues that form the interaction interface differ, suggesting distinct mechanisms. Since the levels of miR16 expression can lead to apoptosis of cancerous cells and prevent cell proliferation, the results of our study provide important clues in understanding how the NCL-miR-MP complex can be targeted in drug design for cancer therapy.