Characterization of Diverse Functions of NRF1 Nuclear Localization Sequence

Most of the transcription factors require a nuclear localization signal (NLS) to translocate to the nucleus via the nuclear import machinery. Initially, classical monopartite NLS with an arginine-lysine basic box was discovered, later less common bipartite NLS with two basic boxes separated with a spacer was found in certain nuclear proteins. Although both types of signals perform the same nuclear translocation function, it is not clear what role does the spacer play in the translocation and what benefits do two basic boxes give to a protein. We studied NRF1 - a transcription factor with a bipartite NLS. We dissected the function of the NLS of NRF1 and found that the spacer plays an auxiliary role in the transcriptional activity of NRF1, and phosphorylation inside the spacer regulates the nuclear translocation of NRF1 when combined with the posttranslational modifications of the NLS. We discovered that the spacer of NRF1 is not necessary for the translocation but it is indispensable for the transcriptional activation. We found that only a single basic box at the N-terminal of NLS is necessary for the nuclear translocation, while C-terminal basic box deletion shows no effect on the NRF1 subcellular localization. Additionally, we found a new downstream target of NRF1-ZNF777.