RIP-chip analysis of Tino/hMEX-3D mRNPs

Tino is an A+U-Rich Element (ARE) binding protein first identified through its ability to bind to bcl-2 mRNA and to contribute to its degradation. It has recently been recognized as a shorter form of the human Mex-3D protein (hMex-3D), one of the four members of the family of Mex-3 RNA-binding phosphoproteins. In C. elegans, ceMex-3 is a translational regulator that plays a key role in early embryonic development and in the maintenance of worm germ line totipotency. To examine the potential functional conservation between ceMex-3 and hMex3, we have used complementary microarray-based approaches to identify mRNAs directly bound to Tino/hMex-3D. Computational analysis of these target mRNAs resulted in the identification of an U-rich, 34- to 39-nucleotide long, consensus, forming loops of variable sizes. Remarkably, more than half of Tino/hMex-3D targets also contain the consensus for Quaking, which is the human ortholog of GLD-1, a regulator of nematode gametogenesis. All together, our results suggest that Tino/hMex-3D belongs to a regulatory circuit of mRNA trans-acting factors involved in cell fate and differentiation. Keywords: RIP-chip and (recombinant)RIP-Chip analysis of Tino/hMEX-3D mRNPs To identify the transcripts directly bound in vivo by Tino/hMex-3D, we have used two complementary strategies. First, using Tino-his transfected HEK293 cells, we have analysed the RNAs immunoprecipitated by Tino/hMex-3D (RNA-IP complexes). Second, we have developed an in vitro nitrocellulose RNA-protein binding assay using a truncated form of Tino, TinoΔRING-his. First for the in vivo approach, we have transiently transfected HEK293 cells with a His-tagged Tino expressing construct (see Materials) and isolated Tino target mRNAs by immunoprecipitation (IP) assays carried out under conditions preserving mRNA-protein complex integrity. Second, for the in vitro approach, cRNAs were prepared from a human placenta cDNA library and incubated with TinoΔRING-his. This deleted form of Tino-his protein conserves its mRNA binding activity. mRNAs directly bound to TinoΔRING-his protein were subsequently isolated using a nitrocellulose RNA-protein binding assay. A supplementary file of ordered genes based on z-ratios, derived from experimental and negative control samples for each Affymetrix probe, is appended below.