Throughout rat brain development, expression of histones variants is mainly regulated at the post-transcriptionallevel. We previously cloned two cDNAs encoding, respectively, PIPPin (or CSD-C2), a brain-enriched protein able tobind the 3’end of H1° and H3.3 mRNAs, and LPI (longer isoform of PEP-19). Both PEP-19 and LPI are brain-specific. Bywestern blot, we found that PIPPin expression in PC12 cells is enhanced by NGF-induced differentiation. Weinvestigated 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 whethercalmodulin could interfere with RNA-binding, and found that calmodulin competes with H1° RNA binding to bothproteins, while it is not able to bind RNA on its own. This finding suggests that, in the brain, PEP19 and LPI couldinduce histone mRNA translation in response to calcium. Using biotinylated H1°/H3.3 RNA, we isolated from rat brainby chromatography a group of proteins which were analyzed by mass spectrometry. Among them someheterogeneous nuclear ribonucleoproteins (HnRNP K, A1, A2/B1) and the Hsc70 chaperone. By western blot of thepurified fraction, we also evidenced PIPPin. We are currently studying the interactions among these proteins by coimmunoprecipitationassays. Finally, by using recombinant PIPPin as a bait, we isolated a group of its interactors whichare now under investigation.
|Numero di pagine||2|
|Stato di pubblicazione||Published - 2012|