Short-lived AUF1 p42-binding mRNAs of RANKL and BCL6 have two distinct instability elements each.

Abstract

Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH/3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, numerous mRNAs were enriched without a high ARE score. The enrichment of tetrameric and pentameric sequences suggests a broad AUF1 p42-binding spectrum at short U-rich sequences flanked by A or G. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3'UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure.