Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling

Oak D Jo,Jheralyn Martin,Andrew Bernath,Janine Masri,Alan Lichtenstein,Joseph Gera

doi:10.1074/jbc.m801185200

Abstract

The translation of the cyclin D1 and c-myc mRNAs occurs via internal ribosome entry site (IRES)-mediated initiation under conditions of reduced eIF-4F complex formation and Akt activity. Here we identify hnRNP A1 as an IRES trans-acting factor that regulates cyclin D1 and c-myc IRES activity, depending on the Akt status of the cell. hnRNP A1 binds both IRESs in vitro and in intact cells and enhances in vitro IRES-dependent reporter expression. Akt regulates this IRES activity by inducing phosphorylation of hnRNP A1 on serine 199. Serine 199-phosphorylated hnRNP A1 binds to the IRESs normally but is unable to support IRES activity in vitro. Reducing expression levels of hnRNP A1 or overexpressing a dominant negative version of the protein markedly inhibits rapamycin-stimulated IRES activity in cells and correlated with redistribution of cyclin D1 and c-myc transcripts from heavy polysomes to monosomes. Importantly, knockdown of hnRNP A1 also renders quiescent Akt-containing cells sensitive to rapamycin-induced G(1) arrest. These results support a role for hnRNP A1 in mediating rapamycin-induced alterations of cyclin D1 and c-myc IRES activity in an Akt-dependent manner and provide the first direct link between Akt and the regulation of IRES activity.

Highlights

A majority of eukaryotic mRNAs contain 5Ј-UTRs2 that are relatively unstructured and typically less than 100 nucleotides in length, which allows for efficient cap-dependent translation initiation
Identification of hnRNP A1 as a Cyclin D1 and c-myc 5Ј-UTRbinding Protein—Since we had previously demonstrated that the 5Ј-UTRs of the cyclin D1 and c-myc mRNAs possessed Akt-dependent internal ribosome entry site (IRES) activity, we initially sought to identify which parts of the leader sequences were responsible for these effects
We generated deletion constructs of both the human cyclin D1 and c-myc 5Ј-UTRs and tested whether the dicistronic reporter mRNAs demonstrated Akt-dependent IRES activity following rapamycin exposure by transient transfection of PTENϪ/Ϫ or PTENϩ/ϩ MEFs. These constructs contained either cyclin D1 or c-myc mRNA leader sequences inserted within the intercistronic region of the plasmid pRF, and translation of the downstream firefly luciferase cistron occurred via internal ribosome entry (Fig. 1A)

Summary

Introduction

A majority of eukaryotic mRNAs contain 5Ј-UTRs2 that are relatively unstructured and typically less than 100 nucleotides in length, which allows for efficient cap-dependent translation initiation. Several proteins that regulate IRES activity, collectively termed IRES trans-acting factors (ITAFs), have been described [7] These ITAFs function by associating with the IRES and either facilitate direct ribosome binding with the mRNA or alter the structure of the IRES. The activation of cap-dependent protein synthesis by Akt is phylogenetically conserved and Akt is known to directly activate a number of canonical translation initiation factors and stimulate ribosome biogenesis [23]. Based on our previous studies, which implicated Akt signaling in the regulation of constitutive and rapamycin-stimulated cyclin D1 and c-myc IRES activity [8], we have investigated the mechanism by which this control takes place for these IRESs

Methods

Results

Conclusion