Functional Genomics Evidence Unearths New Moonlighting Roles of Outer Ring Coat Nucleoporins

There is growing evidence for the involvement of Y-complex nucleoporins (Y-Nups) in cellular processes beyond the inner core of nuclear pores of eukaryotes. To comprehensively assess the range of possible functions of Y-Nups, we delimit their structural and functional properties by high-specificity sequence profiles and tissue-specific expression patterns. Our analysis establishes the presence of Y-Nups across eukaryotes with novel composite domain architectures, supporting new moonlighting functions in DNA repair, RNA processing, signaling and mitotic control. Y-Nups associated with a select subset of the discovered domains are found to be under tight coordinated regulation across diverse human and mouse cell types and tissues, strongly implying that they function in conjunction with the nuclear pore. Collectively, our results unearth an expanded network of Y-Nup interactions, thus supporting the emerging view of the Y-complex as a dynamic protein assembly with diverse functional roles in the cell.


NUP98 [FBpp0083851]
Nup98 (Nup98-96) -encoded in D. melanogaster by the CG10198 gene -is involved in lethal hybrid incompatibility, like Nup160, between D. melanogaster and D. simulans 2 . The largest Nup98 transcript (Nup98-RA) encodes a single, large precursor polyprotein of 1960 residues that, in other eukaryotes, and thus presumably in flies, autoproteolytically cleaves itself yielding separate Nup98 and Nup96 proteins (Nup145N and Nup145C respectively in yeast), with only Nup96/ Nup145-C being a stable member of the Y-complex 14 .
The full-length 1960-residue sequence identifies succinctly its shortest isoform of the auto-proteolytic product as well as multiple variants of longer length, primarily from lower organisms including fungi (autoproteolytic autopeptidase Pfam_04096). Despite its wide distribution across eukaryotes, Nup98 (Nup98-96) is a conserved, well-defined domain represented by either the long form (around 2000 residues) or the short form (around 1000 residues). The N-terminus contains multiple FG repeats and regions common with other nucleoporins, evidently including orthologs SONB/ Nup189 (Data Supplement DS09). The strongly conserved sequence features of Nup98 characterize this well-defined protein family that can be readily detected: with a limit set at 500 hits, the lowest sequence similarity level observed is 24% identity.  Table 2). The region 1314-1769 matches proteins of known structure (e.g. 3IKO), which include the complex with SEC13 16 . In total, there are >500 sequences detected at iteration 10, without false positives and a very deep phylogenetic distribution across the eukaryotes (Supplementary Table 1).

NUP75 [FBpp0085954]
Nup75 -encoded in D. melanogaster by the CG5733 gene -corresponds to a 668 residue long protein. Nup75 (Nup85 in yeast) shares the same α-helical fold with another two members of the group (Nup107/ScNup84, and Nup96/ ScNup145C) also found in Nic96 and interestingly the COPII coatomer element Sec31, suggesting an evolutionary link between NPCs and COP/clathrin-like membrane coats 13,17,18 . Structural homologs are co-crystallized with S. cerevisiae Seh-1 13,18 . The length variation of the archetypal 668-residue protein is due to a 300-residue long N-terminal extension in a significant majority of fungal proteins. Iteration 4 yields 314 homologs (Pfam_07575) (Supplementary Table 1). The family exhibits wide distribution across eukaryotes as previously demonstrated, including amoebozoa and Bacillariophyta (stramenopiles) e.g. Thallassiosira pseudonana CCMP1335 (GI:223997390) 19 . Curiously, one puzzling hit is from the Nup98 family in Harpegnathos saltator (GI:307191801) 20 (Data Supplement DS09). However, there are five cases of length variation not detected previously, which correspond to possible domain involvement of Nup75 into other associations (Supplementary Table 2). is also homologous to Nup75 22 ; the C-terminus of this protein, more than 1000-residues long, contains two instances of the SMC domain (structural maintenance of chromosomes/condensins). [Case 19:] Fifth, the 1828-residue long Vitamin h transporter 1 from Grosmannia clavigera N-terminus (residues 400-1027, up to 1260) (GI:320590333) 23 also exhibits the presence of Nup75; the remainder of this protein contains a MFS (major facilitator superfamily) domain (residues ~1581-1678) involved in secondary transport. Note that none of these domain associations of Nup75 do not appear to be supported by nucleotide sequence searches (see Methods and Supplementary Table 2).

NUP43 [FBpp0082998]
Nup43 and Nup37 were initially identified only in vertebrates and are absent from the S. cerevisiae complex 24,25 . The 358residue Nup43 -encoded in D. melanogaster by the CG7671 gene -contains WD40 repeats in positions 88-358. Recently, the three-dimensional structure of human Nup43 was determined (PDB identifier: 4I79, unpublished). The N-terminal domain specifically recognizes 102 homologs by iteration 4 (Supplementary Table 1) including Drosophila and other insects, lower and then higher vertebrates, and echinozoa (GI:115623733, hypothetical protein is the sea urchin Nup43 ortholog). It is interesting to note that Nup43 is detected in fungi for the first time 26 , in the species Batrachochytrium dendrobatidis JAM81 BATDEDRAFT_37327 (GI:328767770), 379 residues long. No other 'orthologs' in fungi can be detected. The significance of this taxonomic anomaly is not understood at present; the closest relatives of this protein are Drosophila Nup43s, possibly due to sampling/over-representation at this taxonomic range. Despite claims to the contrary 27 , we were unable to establish homology between the masked Nup43 query sequence against a number of plant proteins previously characterized as Nup43 orthologs e.g. in Medicago truncatula (GI:355511317); instead, all these proteins contain WD40 repeats and in some cases are annotated as such. Curiously, the hamster sequence is only 161-residue long (GI: 344241050); the reported missing region is also absent from certain mammalian isoforms. The human sequence (GI: 55663479) contains a 61-residue N-terminal domain of unknown nature, also found in other related species. A sequence from sea urchin Strongylocentrotus purpuratus (GI:115623733) included a 79-residue direct repeat (118-206) 100% identical with an adjacent sequence (227-305) -later updated to a corrected version (GI:390334509).
[Case 20:] The search also returns an Ascaris suum (nematode) MIF 448-residue protein (GI:324503956) 28 , appearing to contain a macrophage migration inhibitory factor (MIF) domain 29 at its N-terminus (positions 2-94) and the Nup43 region at the C-terminus (positions 115-174). As this finding is not supported in related species or across databases -in fact, in the same species there is a 115-residue protein (GI:49257069) without the Nup43 domain -while the 2976-bp sequence was generated by a shotgun/assembly project, it is doubtful that this association of MIF with Nup43 reflects reality and is likely to be a short-read/assembly artifact, not supported by nucleotide searches (Supplementary Table  2).
[Case 21:] Surprisingly, Nup43 is found at the N-terminal region of 1000-residue long proteins of six Apocrita insects with DHX15 helicase at the remaining sequence (Supplementary Table 2 . This region has not been recognized as unique to the Nup43 family and was generally annotated as a WD40 repeat region. We unequivocally demonstrate that the DHX domain is present in these insect Nup43s, while missing from the Drosophila Nup43s. These organisms do not contain another copy of Nup43; thus the N-terminus of these genes can be considered as the Nup43 ortholog in these species. Gene CG11107 in D. melanogaster and its orthologs in Drosophila and other insect species is reciprocally the DHX15 ortholog of the above six proteins, thus implying that it might be functionally associated with Nup43 30 . The recent immunoisolation of a Werner Helicase interacting protein (WHIP) with the human Y-complex also points towards a potential functional link between the Y-complex and helicases 31 .

SEH1 [FBpp0087938]
SEH1 -encoded in D. melanogaster by the CG8722 gene -also known as nup44A from its chromosomal location, is a central but least stable component of the Y-complex 25 , also being part of the SEA complex 35 . However, it confers compositional integrity to the complex as depletion by RNA interference of this single subunit depletes the rest of the subunits from kinetochores 36 . The 356-residue SEH1 contains WD40 repeats throughout its length resulting in an open six-bladed β-propeller, with a seventh blade provided by its partner Nup85/75 13 . Following CAST masking (score≥10; instead of 15), during iteration 1 the search already returns SEH1 homologs as well as their closest relatives, those in SEC13 family. Despite the WD40 repeat structure, the search identifies the bona fide homologs with specificity (Supplementary Table 1). [Case 22:] The only notable exception is a 794-residue phosphoglucomutase from Brugia malayi 37 , which contains SEH1/SEC13 repeats at its N-terminus (residues 1-347) and the enzyme domain at the C-terminus (398-794C). It should be noted that B. malayi has another gene coding for SEC13 (GI:170588105), thus excluding the possibility that the PGM gene is a sequencing artifact. In total, there are (at least) 500 sequences detected at iteration 6 (including 7 proteins of known structure), without false positives (Data Supplement DS09).  Table 1, Data Supplement DS12, Supplementary Table 2). As SEH1 and SEC13 identify each other, all observations for SEH1 are also valid for SEC13 (see text for details).     Figure 3B with correlation values). Color scheme: Dark green -self-comparison of Y-Nups; Light green -cross-comparison of Y-Nups; Yellow -marks most distant Y-Nup query in cross-comparison, serves as threshold for bottom section of table i.e. domain associations; Grey -query ranks above threshold except domain-supported cases; White -query ranks below threshold, not considered further; Blue shades signify levels of support by expression for query: petrol blue -supported as ranking above threshold for human ortholog, sky blue -supported as ranking above threshold for all paralogs, ice blue -not supported as ranking below threshold (grey font) or above threshold (purple font) for one paralog only. For details, see Methods.