Identification of RNA-binding Proteins That Partner with Lin28a to Regulate Dnmt3a Expression

Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that Lin28a activity is dependent on many proteins, including three helicases and four RNA-binding proteins. The suppression of four of these proteins, namely Ddx3x, Hnrnph1, Hnrnpu or Syncrip, interferes with the binding of Lin28a to the Dnmt3a mRNA, thus suggesting that they are part of an oligomeric ribonucleoprotein complex that is necessary for Lin28a activity.