Poster abstracts
Poster number 61 submitted by Amra Ismail
Investigation of IRES-mediated Translation of PUMA mRNA, Initiation Factor Requirements and Search for IRES-Trans Acting Factors (ITAFs)
AMRA ISMAIL (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University), Shuai Zhao (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University), Crystal M. Weyman (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University), Anton A. Komar (Department of Biological, Geological and Environmental Sciences and the Center for Gene Regulation in Health and Disease, Cleveland State University)
Abstract:
Canonical translation initiation in majority of cellular mRNAs occurs by a cap-dependent/scanning mechanism, whereby the 43S preinitiation complex binds to the mRNA 5’ terminal cap structure and scans the 5’UTR of mRNA in search of the initiation codon. Internal Ribosome Entry Sites (IRESs) are cis-acting elements, located in the 5’UTRs of some viral and cellular mRNAs that facilitate direct recruitment of the 40S ribosomal subunits near the AUG codon. IRES-mediated translation initiation may proceed without the help of many canonical initiation factors but may require additional IRES-trans-acting factors (ITAFs).
The proapoptotic Bcl-2 family member PUMA has been previously shown to contain an IRES element that is active under conditions of eIF2-α phosphorylation and hypophosphorylation of eIF4E-BP that inhibits cap-dependent translation.
We further investigated the mechanism for PUMA mRNA recruitment to the ribosome and found that unlike class III or IV viral IRESs, PUMA IRES is not able to bind 40S ribosomal subunits directly. We also analyzed PUMA IRES initiation factor requirements and found that PUMA IRES requires intact eIF4G and eIF4A for its activity, which is reminiscent of the hepatitis E virus (HEV) IRES requirements. RNA affinity pull down assays and mass spectrometric analysis identified Hsp70 as one of the proteins that binds PUMA IRES with high affinity. Gel shift assays confirmed that the binding is specific. Further in vitro studies reveal that IRES-mediated translation of PUMA mRNA is enhanced in the presence of Hsp70 protein.
Keywords: Internal Ribosome Entry Sites, ITAF (IRES-trans acting factors)