Date of Degree

2-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Shaneen Singh

Committee Members

Anjana Saxena

Jayne Raper

Luis Quadri

Shubha Govind

Dario Palmieri

Subject Categories

Biochemistry | Bioinformatics | Structural Biology

Keywords

RNA binding protein, nucleolin, miRNA, mRNA UTR, molecular modeling, molecular docking

Abstract

Nucleolin (NCL) is a stress responsive multifunctional nucleolar protein and accounts for 10% of the total nucleolar protein content. NCL belongs to the class of RNA binding proteins (RBPs) that regulate many important cellular processes through their interactions with different RNA molecules. The dysregulation of RBPs and the RNA metabolism pathways they intersect is a known driver of tumorigenesis. NCL regulates ribosome biogenesis, chromatin remodeling, microRNA processing, and gene expression on multiple levels. The RNA-protein interactions of NCL are primarily driven by its four RNA binding domains (RBDs). NCL is known to interact with a growing list of primary-miRNA (pri-miRNA) molecules which lead to breast cancer progression and poor disease prognosis. NCL is also known to interact with both 3’ and 5’ untranslated region of a group of mRNA molecules, similarly, involved in tumorigenesis and poor disease outcome. Mechanisms driving NCL-RNA interactions in the context of human cancers are currently unknown and this gap in knowledge is further compounded by the incomplete state of structural information for NCL. In this study, we analyzed NCL-miRNA and NCL-mRNA interactions in depth with a focus on a subset of RNA molecules that are implicated in cancer. The lack of structural information for NCL RBD1-4, pri-miRNA, and mRNA UTR molecules was addressed in this study by the generation of robust 3D models. Interactions of NCL RBDs with specific RNA molecules were investigated using RNA-protein docking algorithms. These analyses have revealed a comprehensive map of the NCL-RNA interface for individual RNA pri-miRNA and mRNA UTRs as well as overall trends in recognition of these RNA species by NCL. Our results indicate that NCL RBDs 3 and 4 preferentially interact with miRNA molecules whereas a collaboration between RBDs 1 and 2 and RBDs 3 and 4 is predicted to drive most NCL-mRNA interactions. We also investigated the oligomerization modes of NCL since oligomerization is known to regulate certain important RNA binding functions of NCL. Our results revealed that NCL was predicted to adopt multiple oligomerization modes and each one was predicted to have different RNA/protein binding capabilities. This theoretical study provides critical clues on mechanisms driving NCL RBD-mRNA/miRNA target specificity. The in silico predictions derived from this study further our long-term goal of elucidating the detailed mechanisms of NCL-RNA interactions in cancer by providing a rational setup for experimental validation of the proposed mechanism of interaction.

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