S1‐1/RBM10: Multiplicity and cooperativity of nuclear localisation domains

Abstract

S1-1, also called RBM10, is an RNA-binding protein of 852 residues. An alteration of its activity causes TARP syndrome, a severe X-linked disorder with pre- or post-natal lethality in affected males. Its molecular function, although still largely unknown, has been suggested to be transcription and alternative splicing. In fact, S1-1 localises in the nucleus in tissue cells and cultured cells. By deletion and substitution mutagenesis, a classical 17-amino-acid (aa) nuclear localisation sequence (NLS1) was identified at aa 743-759 in the C-terminal region of S1-1. NLS1 was bipartite, with its N-terminal basic cluster weakly contributing to the NLS activity. S1-1 contained two additional NLSs. One was in the aa 60-136 RNA recognition motif region (NLS2), and the other was a novel NLS motif sequence in the aa 481-540 octamer-repeat (OCRE) region (NLS3). The OCRE is a domain known to be critical in splicing regulation, as shown with RBM5, a close homologue of RBM10 [Bonnal et al. (2008) Mol. Cell 32, 81-95]. The NLS activities were verified by expressing each DNA sequence linked to EGFP or a FLAG tag. These multiple NLSs acted cooperatively, and S1-1 became completely cytoplasmic after the concomitant removal of all NLS domains. In some cell types, however, S1-1 was partly cytoplasmic, suggesting that cellular localisation of S1-1 is subjected to regulation. The present results indicate that S1-1 contains multiple NLSs that act cooperatively. Among them, the OCRE is a hitherto unreported NLS. The nuclear localisation of S1-1 appears to be regulated under certain circumstances. We discuss these NLSs in relation to the biochemical processes they are involved in.