Rat PIPPin is probably part of a large complex of RNA-binding proteins

Throughout rat brain development, expression of histones variants is mainly regulated at the post-transcriptional level. We previously cloned two cDNAs encoding, respectively, PIPPin (or CSD-C2), a brain-enriched protein able to bind the 3’end of H1° and H3.3 mRNAs, and LPI (longer isoform of PEP-19). Both PEP-19 and LPI are brain-specific. By western blot, we found that PIPPin expression in PC12 cells is enhanced by NGF-induced differentiation. We investigated the RNA-binding properties of the three proteins using their 6 histidine-tagged recombinant fusions and found that they all bind H1° and H3.3 RNAs. Since PEP-19 and LPI are camstatins, we also analyzed whether calmodulin could interfere with RNA-binding, and found that calmodulin competes with H1° RNA binding to both proteins, while it is not able to bind RNA on its own. This finding suggests that, in the brain, PEP19 and LPI could induce histone mRNA translation in response to calcium. Using biotinylated H1°/H3.3 RNA, we isolated from rat brain by chromatography a group of proteins which were analyzed by mass spectrometry. Among them some heterogeneous nuclear ribonucleoproteins (HnRNP K, A1, A2/B1) and the Hsc70 chaperone. By western blot of the purified fraction, we also evidenced PIPPin. We are currently studying the interactions among these proteins by coimmunoprecipitation assays. Finally, by using recombinant PIPPin as a bait, we isolated a group of its interactors which are now under investigation.