The regulation of microRNA biogenesis by ribosome-interacting proteins

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