Identification of the binding proteins of the 3' untranslated region of mouse Ard1b that may be responsible for its translational delay during spermatogenesis

Abstract

Ard1b encodes the catalytic subunit of N(alpha)-acetyltransferase which is believed to play a role in spermatogenesis. In developing mouse testes, the Ard1b transcript level steadily increases before the appearance of Ard1b proteins. Therefore, translation of Ard1b is uncoupled from transcription. The Ard1b gene contains a long 3' untranslated region (UTR). Regulatory elements affecting the stability, localization and translational efficiency of RNA transcripts tend to localize on the 3'UTR of genes. We hypothesized that protein factors are involved in delaying Ard1b translation through their physical interaction with specific sequence motifs present in the 3'UTR of Ard1b transcripts. Our objective was to identify the proteins that bind to the Ard1b 3' UTR. Laboratory Study. The Ard1b 3'UTR was cloned into pCR4-TOPO vector, which was linearized for in vitro transcription in the presence of biotinylated ATP. The biotinylated Ard1b 3'UTR RNA was immobilized on streptavidin-coated magnetic agarose beads and the complex was incubated with mouse testicular lysate. Proteins captured by the Ard1b 3'UTR were subsequently eluted, separated by gel electrophoresis and analyzed by mass spectrometry (MS). The involvement of the candidate proteins in translational repression will be tested in a reporter assay. Three independent pull-down experiments were performed. Multiple proteins were captured by the Ard1b 3'UTR. Among them, 5 proteins (∼21, ∼33, ∼34, ∼35 and ∼ 97 kDa) were consistently revealed on protein gels. Of these, one was identified by MS as the heterogeneous nuclear ribonucleoprotein (hnRNP) C. These results suggest hnRNP C is a candidate repressor of Ard1b translation. Further studies will test if the 15-LOX-DICE regulatory element, located in the Ard1b 3'UTR, might bind hnRNP C and contribute to the delayed translation of Ard1b.