Nuclear protein import, including that of transcription factors such as REST4 or REST/NRSF, is a key control point in regulating gene expression. We previously reported that the proper zinc finger structure of the transcriptional regulator REST4 not only functions in DNA recognition but also is involved in protein-protein interaction and in determination of subcellular localization (26
). It was suggested that zinc finger domains 2 to 5 are important for nuclear localization. Using these zinc finger domains (amino acids 213 to 321) as bait, a yeast two-hybrid assay screen led to the cloning of a novel protein that we have named RILP.
A conserved-domain search analysis showed that RILP has three LIM domains at its N-terminal region. A LIM domain is a cysteine-histidine-rich, zinc-coordinating domain, consisting of tandemly repeated zinc fingers (8
) that are structurally similar to the GATA-type zinc fingers (20
). LIM domains have been found to interact specifically with other LIM proteins (9
), as well as with a variety of other known proteins including basic helix-loop-helix proteins (10
), cytoskeletal components (3
), the insulin receptor (30
), and POU-HD proteins (5
). Since LIM domains are thought to function as protein interaction modules, it is likely that RILP interacts with REST/NRSF and REST4 through its LIM domains. Some proteins that are localized in the nucleus consist primarily of LIM domains, and were therefore named LIM-only proteins (LMO) (22
). However, there appear to be no reports that the LIM domains of mammalian LIM domain-containing proteins are involved in DNA binding per se (4
Coimmunoprecipitation experiments, as well as colocalization of recombinant-tagged RILP and REST4 or REST/NRSF in HEK293 cells, document that RILP interacts with the transcriptional repressor REST/NRSF and the transcriptional regulator REST4. That this interaction is involved in translocation to the nucleus is demonstrated by the finding that reduction of endogenous RILP by siRNA causes the mislocalization of REST4 and REST/NRSF from the nucleus to the cytosol.
The Cys-Ile-Ile-Ser sequence at the C terminus of RILP is possibly a CAAX-type prenylation signal (12
), suggesting that RILP may be farnesylated (because of the serine at X) and thus becomes a membrane-associated protein, such as Ras and Lamin. Indeed, in vitro farnesylation analysis suggested that RILP was farnesylated at the C terminus (M. Shimojo and L. Hersh, unpublished data). In the case of lamin B, which is the major constituent of the lamina, its prenylation appears to be involved in membrane targeting, proper nuclear localization, and protein-protein interactions, which are important for signal transduction. That the CAAX motif of RILP is functional is shown by the mislocalization of a mutant RILP in which this motif was deleted. The cytosolic localization of this mutant RILP suggests that prenylation plays a role in RILP associating with the nuclear membrane. The sensitivity of RILP on intact nuclei to protease digestion indicates that some if not all of RILP is localized to the outer nuclear membrane. This suggests that RILP is involved in the translocation of REST/NRSF and REST4 into the nucleus. For nuclear protein import, it is suggested that most nuclear localization signals (NLSs) are recognized by and form a complex with importin/karyopherin. There are three putative NLSs at residues 617 to 623 (PVLRRSK), resdues 673 to 677 (HRRRR), and residues 818 to 821 (KKKK), which were predicted by the PSORT computer program. Deletion and mutation of each NLS caused mislocalization of RILP to the cytosol (unpublished data), suggesting the possibility that importin/karyopherin may be important for RILP nuclear targeting.
Taken together, these data suggest that RILP functions in the trafficking of the transcriptional repressor REST/NRSF and the transcription regulator REST4 into the nucleus. The data support a mechanism in which RILP is bound to the nuclear membrane through its CAAX motif and binds REST/NRSF and REST4, probably through its LIM domains. We therefore suggest that RILP serves as a nuclear receptor for REST/NRSF, REST4, and probably other nuclear proteins, and is intimately involved in their nuclear targeting.