ROLE OF RNA BINDING PROTEIN IN THE NERVE CELL DIFFERENTIATION

Synthesis of H1˚ and H3.3 histone proteins, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins (RBPs), we have been searching for RBPs involved in the post-transcriptional regulation of the H1˚ and H3.3 genes. Previously, we reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). We showed that LPI, as well as PEP-19, can bind H1˚ RNA. Since PEP19 and LPI contain a calmodulin binding domain, we also investigated whether their ability to bind RNA is affected by calmodulin. Our results show that calmodulin interferes with binding of H1˚ RNA to both PEP-19 and LPI, while it is not able to bind RNA on its own. Pep-19/calmodulin high affinity binding has been demonstrated by Biolayer interferometry (BLI). This finding suggests that calcium/calmodulin may have a role in controlling H1˚ mRNA metabolism in the developing brain. Moreover, in order to improve production of functional LPI/PEP-19, we modified the protocol normally adopted for preparing histidine tagged-proteins from bacteria, by adding an additional purification step. Furthermore, we found that both LPI and PEP-19 can compete for H1˚ RNA binding with PIPPin (also known as CSD-C2), another RBP previously discovered in our laboratory. PIPPin/CSD-C2 binds with high specificity to the mRNAs encoding H1° and H3.3 histone variants, undergoes thyroid hormone-dependent SUMOylation, and has been recently demonstrated to interact with other RBPs. PIPPin belongs to the CSD-containing class of RBPs, also called Y-box proteins, that play a key role in controlling the recruitment of mRNAs to the translational machinery, in response to environmental cues, both in development and in differentiated cells. Another aspect of this study was to confirm histone mRNAs-PIPPin interactions and to describe binding properties through streptavidin-biotin conjugation method, by BLI. We report the data obtained in the case of H3.3 and H1° mRNA-PIPPin interactions, and the specific affinity constant for these bindings. In order to identify RNA portions involved in binding, we used different RNA probes for H3.3 and H1°. In summary, we were able to confirm that PIPPin binds H3.3 and H1° mRNA with very high affinity. Searching for other RBPs, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of CSD-C2. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70, whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70.