Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

Ho-Hyung Woo,Terri Baker,Csaba Laszlo,Setsuko K Chambers

doi:10.1074/mcp.m112.025288

Abstract

CSF-1 mRNA 3'UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3'UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3'UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of nucleolin's actions on CSF-1 mRNA and describe the dependence of miR-130a- and miR-301a-directed CSF-1 mRNA decay and inhibition of ovarian cancer cell motility on nucleolin.

Highlights

MRNA degradation and translation are intimately coupled processes in which RNA-binding proteins (RBPs)1 and miRNAs interact with cis-acting regulatory elements in mRNA-untranslated regions (UTRs). miRNAs usually elicit translation repression and mRNA decay [1,2,3,4], a complex process that involves multiple components, including argonaute (Ago) proteins
To obtain insight into the mechanism by which these cis-acting regulatory elements regulate the expression of CSF-1 in ovarian cancer cells, we generated constructs of luciferase RNA fused to CSF-1 mRNA 3ЈUTR with point mutations in the common miRNA target and/or deletion of Gquadruplex and/or AU-rich elements (AREs) to disrupt the function of these elements (Fig. 1B)
Ago2, and poly(A)-binding protein C (PABPC) May Exist Together as a Complex Containing CSF-1 mRNA in Ovarian Cancer Cells, and Nucleolin Interacts with Ago2 Indirectly via RNA—Because both the G-quadruplex and common miRNA target sequence are in close proximity (Fig. 1A) and nucleolin is required for miR-130a and miR-301a activities on CSF-1 mRNA (Fig. 5), the association of nucleolin with Ago2 as part of the miRNA-induced silencing complex (miRISC) complex was determined by a co-IP assay

Summary

Introduction

MRNA degradation and translation are intimately coupled processes in which RNA-binding proteins (RBPs)1 and miRNAs interact with cis-acting regulatory elements in mRNA-untranslated regions (UTRs). miRNAs usually elicit translation repression and mRNA decay [1,2,3,4], a complex process that involves multiple components, including argonaute (Ago) proteins. To obtain insight into the mechanism by which these cis-acting regulatory elements regulate the expression of CSF-1 in ovarian cancer cells, we generated constructs of luciferase RNA fused to CSF-1 mRNA 3ЈUTR with point mutations in the common miRNA target and/or deletion of Gquadruplex and/or AREs to disrupt the function of these elements (Fig. 1B).

Results

Conclusion