An E2F/miR-20a Autoregulatory Feedback Loop

Yannick Sylvestre,Vincent De Guire,Emmanuelle Querido,Utpal K Mukhopadhyay,Véronique Bourdeau,François Major,Gerardo Ferbeyre,Pascal Chartrand

doi:10.1074/jbc.m608939200

Abstract

The E2F family of transcription factors is essential in the regulation of the cell cycle and apoptosis. While the activity of E2F1-3 is tightly controlled by the retinoblastoma family of proteins, the expression of these factors is also regulated at the level of transcription, post-translational modifications and protein stability. Recently, a new level of regulation of E2Fs has been identified, where micro-RNAs (miRNAs) from the mir-17-92 cluster influence the translation of the E2F1 mRNA. We now report that miR-20a, a member of the mir-17-92 cluster, modulates the translation of the E2F2 and E2F3 mRNAs via binding sites in their 3'-untranslated region. We also found that the endogenous E2F1, E2F2, and E2F3 directly bind the promoter of the mir-17-92 cluster activating its transcription, suggesting an autoregulatory feedback loop between E2F factors and miRNAs from the mir-17-92 cluster. Our data also point toward an anti-apoptotic role for miR-20a, since overexpression of this miRNA decreased apoptosis in a prostate cancer cell line, while inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment. This anti-apoptotic role of miR-20a may explain some of the oncogenic capacities of the mir-17-92 cluster. Altogether, these results suggest that the autoregulation between E2F1-3 and miR-20a is important for preventing an abnormal accumulation of E2F1-3 and may play a role in the regulation of cellular proliferation and apoptosis.

Highlights

Activators of cell cycle progression and promote the entry of quiescent cells into S phase [4, 5]
While overexpression of miR-20a decreased apoptosis in a prostate cancer cell line, inhibition of miR-20a by an antisense oligonucleotide resulted in increased cell death after doxorubicin treatment, pointing to a potential anti-apoptotic role for miR-20a
Regulation of E2F1–3 Expression by miR17/miR20 miRNAs— To investigate whether E2F1 expression was regulated by factors binding to its 3Ј-UTR, we fused the intact 3Ј-UTR of E2F1 to a luciferase reporter

Summary

Introduction

Activators of cell cycle progression and promote the entry of quiescent cells into S phase [4, 5]. Both 3Ј-UTRs were cloned at the 3Ј end of the luciferase gene in the pGL3 control plasmid and transfected in HeLa cells (due to its large size, the E2F2 3Ј-UTR that was cloned contained only the first two miR-20a-binding sites).

Results

Conclusion