Ld be rescued by expressing both RCC1 and ELYS fused to DNA binding domains. ELYS can bind 
the H2AH2B dimer, suggesting an interaction with these histones is important in recruiting ELYS to nucleosomes. ELYS also contains a variant type of AThook DNA binding domain, and it had previously been thought that NPC formation is initiated by recruitment of ELYS to DNA. However, biochemical experiments showed that the AT-hook is insufficient for binding to either naked DNA or nucleosomes, although it does contribute to nucleosome interaction. Altogether, although remaining to be clarified by structural analyses, this suggests that interaction with both H2AH2B and DNA within the context of a nucleosome is required for chromatin recruitment of ELYS. Once on chromatin, ELYS induces NPC formation by recruiting the Nup107 complex, an important structural NPC component. The RCC1-dependent and ELYS-dependent NPC assembly branches intersect, and chromatin association of the Nup107 complex requires RanGTP, at least partially explaining the RanGTP requirement in NPC formation . Similarly, addition of RanQ69L to Xenopus egg extracts can increase the chromatin association of ELYS. However, RanGTP may not be absolutely required, as preventing the formation of RanGTP does not interfere with the association of ELYS with chromatin. At present, it is not clear how ELYS association with chromatin can occur independently of RanGTP even though chromatin association of the Nup107 complex requires RanGTP. Fractionation experiments showed ELYS and the Nup107 complex to be associated with each other in the absence of RanGTP and in the presence of importin , suggesting RanGTP not to regulate the interaction between ELYS and the Nup107 complex. Future studies will be needed to resolve this matter. Species-specific differences may also exist, as interference with the Ran-system impedes chromatin association of ELYS in C. elegans. Finally, RanGTP is also involved in later steps in NPC assembly . Whatever the exact function of RanGTP in NPC formation is, it can be bypassed under certain conditions. Fusing ELYS with the INM protein emerin allows NPC formation on nucleosome-free oocyte pronuclei that do not contain RCC1. At first glance, this mechanism may appear to be related to a second pathway for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19853566 NPC assembly, which mediates CF-101 price insertion of NPCs into intact membranes. However, this pathway normally occurs independently of ELYS and requires RanGTP. Therefore, the mechanistic basis of this phenomenon is currently not understood. Altogether, by recruiting membrane components and NPC components of the 
NE, DNA and nucleosomes together specify the assembly of a functional NE. NPCs can assemble both at mitotic exit, concomitantly to NE formation, as well as into an intact envelope. NPC assembly at mitotic exit is much faster, and rapidly dividing cells may thus be more dependent on this pathway. Author get MRT-67307 Manuscript Author Manuscript Author Manuscript Author Manuscript Bioessays. Author manuscript; available in PMC 2016 October 01. Zierhut and Funabiki Page 10 Nuclear pore complex proteins contribute to spindle function Several nucleoporins have secondary functions that connect NPCs with mitotic spindles. Both ELYS and the Nup107 complex can be detected in an interdependent manner at kinetochores and spindle poles, and seem to regulate both kinetochore and spindle function in a species-specific manner. During chromosome-induced spindle assembly in X. laevis egg extracts, immunodeplet.Ld be rescued by expressing both RCC1 and ELYS fused to DNA binding domains. ELYS can bind the H2AH2B dimer, suggesting an interaction with these histones is important in recruiting ELYS to nucleosomes. ELYS also contains a variant type of AThook DNA binding domain, and it had previously been thought that NPC formation is initiated by recruitment of ELYS to DNA. However, biochemical experiments showed that the AT-hook is insufficient for binding to either naked DNA or nucleosomes, although it does contribute to nucleosome interaction. Altogether, although remaining to be clarified by structural analyses, this suggests that interaction with both H2AH2B and DNA within the context of a nucleosome is required for chromatin recruitment of ELYS. Once on chromatin, ELYS induces NPC formation by recruiting the Nup107 complex, an important structural NPC component. The RCC1-dependent and ELYS-dependent NPC assembly branches intersect, and chromatin association of the Nup107 complex requires RanGTP, at least partially explaining the RanGTP requirement in NPC formation . Similarly, addition of RanQ69L to Xenopus egg extracts can increase the chromatin association of ELYS. However, RanGTP may not be absolutely required, as preventing the formation of RanGTP does not interfere with the association of ELYS with chromatin. At present, it is not clear how ELYS association with chromatin can occur independently of RanGTP even though chromatin association of the Nup107 complex requires RanGTP. Fractionation experiments showed ELYS and the Nup107 complex to be associated with each other in the absence of RanGTP and in the presence of importin , suggesting RanGTP not to regulate the interaction between ELYS and the Nup107 complex. Future studies will be needed to resolve this matter. Species-specific differences may also exist, as interference with the Ran-system impedes chromatin association of ELYS in C. elegans. Finally, RanGTP is also involved in later steps in NPC assembly . Whatever the exact function of RanGTP in NPC formation is, it can be bypassed under certain conditions. Fusing ELYS with the INM protein emerin allows NPC formation on nucleosome-free oocyte pronuclei that do not contain RCC1. At first glance, this mechanism may appear to be related to a second pathway for PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19853566 NPC assembly, which mediates insertion of NPCs into intact membranes. However, this pathway normally occurs independently of ELYS and requires RanGTP. Therefore, the mechanistic basis of this phenomenon is currently not understood. Altogether, by recruiting membrane components and NPC components of the NE, DNA and nucleosomes together specify the assembly of a functional NE. NPCs can assemble both at mitotic exit, concomitantly to NE formation, as well as into an intact envelope. NPC assembly at mitotic exit is much faster, and rapidly dividing cells may thus be more dependent on this pathway. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Bioessays. Author manuscript; available in PMC 2016 October 01. Zierhut and Funabiki Page 10 Nuclear pore complex proteins contribute to spindle function Several nucleoporins have secondary functions that connect NPCs with mitotic spindles. Both ELYS and the Nup107 complex can be detected in an interdependent manner at kinetochores and spindle poles, and seem to regulate both kinetochore and spindle function in a species-specific manner. During chromosome-induced spindle assembly in X. laevis egg extracts, immunodeplet.