Mechanism of the Internal Ribosome Entry Site-mediated Translation of Serine Hydroxymethyltransferase 1

Abstract

The 5'-untranslated region (UTR) of serine hydroxymethyltransferase 1 (SHMT1) contains an internal ribosome entry site (IRES) that regulates SHMT1 expression, a rate-limiting enzyme in de novo thymidylate biosynthesis. In this study, we show that the SHMT1 IRES is the first example of a cellular IRES that is poly(A) tail-independent. Interactions between the 5'-UTR and 3'-UTR functionally replaced interactions between the poly(A) tail and the poly(A)-binding protein (PABP) to achieve maximal IRES-mediated translational efficiency. Depletion of the SHMT1 IRES-specific trans-acting factor (ITAF) CUG-binding protein 1 (CUGBP1) from in vitro translation extracts or deletion of the CUGBP1 binding site on the 3'-UTR of the SHMT1 transcript decreased the IRES activity of non-polyadenylylated biscistronic mRNAs relative to polyadenylylated biscistronic mRNAs and resulted in a requirement for PABP. We also identified a novel ITAF, heterogeneous nuclear ribonucleoprotein H2 (hnRNP H2), that stimulates SHMT1 IRES activity by binding to the 5'-UTR of the transcript and interacting with CUGBP1. Collectively, these data support a model for the IRES-mediated translation of SHMT1 whereby the circularization of the mRNA typically provided by the eukaryotic initiation factor (eIF) 4G/PABP/poly(A) tail interaction is achieved instead through the hnRNP H2/CUGBP1-mediated interaction of the 5'- and 3'-UTRs of the SHMT1 transcript. This circularization enhances the IRES activity of SHMT1 by facilitating the recruitment and/or recycling of ribosomal subunits, which bind to the transcript in the middle of the 5'-UTR and migrate to the initiation codon via eIF4A-mediated scanning.