Date of Graduation
5-2014
Document Type
Dissertation (PhD)
Program Affiliation
Cancer Biology
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Dihua Yu, MD, PhD
Committee Member
Ann-Bin Shyu, PhD
Committee Member
Dos Sarbassov, PhD
Committee Member
Min Gyu Lee, PhD
Committee Member
Richard Behringer, PhD
Abstract
MicroRNA (miRNA) are small, non-coding RNAs that affect gene expression through degradation of complementary mRNA targets or inhibition of translation. As they affect approximately 50% of all cellular processes, miRNA are tightly regulated by the cell through transcriptional and post-transcriptional mechanisms. Transcribed miRNA are capped and polyadenylated (referred to as pri-miRNA) which are cleaved by Drosha and DGCR8 to generate 60-90 nucleotide precursor miRNA. The precursors are cleaved again by Dicer and loaded into the RNA-induced silencing complex (RISC) of which Argonaute 2 is the functional component. Many of the proteins involved in miRNA biogenesis share a common role in ribosomal RNA regulation. Here we characterize two ribosome-associated proteins that are important for miRNA biogenesis. In one study, we identified nucleolin as a positive regulator of pri-miR-15a/miR-16-1 biogenesis. Nucleolin expression is inversely proportional to mature miR-15a/miR-16-1 expression. While nuclear localization of nucleolin increases miR-15a/16-1 expression, cytoplasmic localization of nucleolin decreases it in a mechanism dependent on the interaction of nucleolin with Drosha and DGCR8. Furthermore, pri-miR-15a/miR-16-1 is bound by nucleolin, which facilitates its processing in vitro. In another study, we analyzed TCGA patient datasets to uncover a miRNA signature associated with ZEB1/2 expression that refutes current models of miR-200 family (miR-200a/b/c, miR-141, miR-429) regulation. In breast cancer cell lines with low miR-200 expression an abundance of primary and precursor species exist. We found these precursors are able to regulate other miR-200 family members in a coherent feedforward loop, independent of transcription, by titrating away a repressor complex. We identified the repressor as Receptor of Ribosome Binding Protein 1 (RRBP1) by developing a new technique to capture endogenous protein-RNA complexes in vivo called Cross-linking and PNA Pulldown (CLaPP) assay. RRBP1 inversely correlates with miR-200 expression in cell lines and through gain- and loss-of-function studies. TGF-b treatment transcriptionally increased RRBP1 abundance resulting in loss of miR-200 expression. Lastly, RRBP1 was found to directly associate with miR-200 precursors through iCLIP analysis.
In summary, the ribosome-associated proteins nucleolin and RRBP1 were identified and characterized as two novel proteins involved in miRNA biogenesis, each forming feedforward miRNA loops that regulate distinct cellular processes.
Keywords
Nucleolin, RRBP1, p180, ES130, breast cancer, CLaPP