The central transport channel (CTC) of nuclear pore complexes (NPCs) is made up of three nucleoporins Nup62, Nup58 and Nup54. In which manner and capacity, these nucleoporins form the CTC, is not yet clear. We explored the CTC Nups from various species and observed that distinct biochemical characteristics of CTC Nups are evolutionarily conserved. Moreover, comparative biochemical analysis of CTC complexes showed various stoichiometric combinations of Nup62, Nup54 and Nup58 coexisting together. We observed the conserved amino-terminal domain of mammalian Nup93 is crucial for the anchorage of CTC and its localization to NPCs. We could reconstitute and purify mammalian CTCÁNup93 quaternary complex by co-expressing full length or N-terminal domain of Nup93 along with CTC complex. Further, we characterized CTCÁNup93 complex using small angle X-ray scattering and electron microscopy that revealed a "V" shape of CTCÁNup93 complex. Overall, this study demonstrated for the first time evolutionarily conserved plasticity and stoichiometric diversity in CTC Nups. K E Y W O R D S central transport channel, nuclear pore complex, Nup93, small angle X-ray scattering, co-immunoprecipitation Parshuram J. Sonawane and Pravin S. Dewangan are lead co-authors.

This study was aimed at isolation of cells from urine and skin on the ventral part of the tails of healthy adult female buffaloes (Bubalus bubalis), an area rarely exposed to solar radiation, establishment of the cells in culture, and their use as donor cells for production of buffalo embryos by handmade cloning (HMC). The blastocyst rate and total cell number of urine-and tail skin-derived embryos were similar to those of control embryos derived from ear skin cells; however, their apoptotic index was lower ( p < 0.05) than that of control blastocysts. The global level of histone H3 acetylated at lysine 9 (H3K9ac) was similar in the three types of donor cells and in urine-and tail skin-derived HMC blastocysts and in vitro-fertilized (IVF) blastocysts (controls). The global level of histone H3 trimethylated at lysine 27 (H3K27me3) in the cells was in the order ( p < 0.05) urine ‡ tail skin > ear skin-derived cells, whereas in blastocysts, it was higher ( p < 0.05) in urine-and tail skin-derived HMC blastocysts than that in IVF blastocysts. The expression level of CASPASE3, CAS-PASE9, P53, DNMT1, DNMT3a, OCT4, and NANOG, which was similar in HMC blastocysts of three the groups, was lower ( p < 0.05) than that in IVF blastocysts, whereas that of HDAC1 was similar among the four groups. Following transfer of urine-derived embryos (n = 10) to five recipients (two embryos/recipient), one of the recipients delivered a normal calf that is now 5 weeks old.

Following IVF, embryos which cleave early have been shown to have higher developmental competence and quality than those that cleave relatively later across many species. We investigated the effect of time of cleavage on the developmental competence, quality, epigenetic status and gene expression in buffalo embryos produced by handmade cloning (HMC). Following classification of embryos as early cleaving (EC) or late cleaving (LC) based on whether they had cleaved or not at 24 h post in vitro culture, 54% (164/303) were found to be EC and the rest to be LC. The blastocyst rate (58.1 ± 3.4 vs 36.9 ± 1.6%, p < 0.01) and the total cell number (285.5 ± 41.9 vs 141.4 ± 36.1, p < 0.05) were higher, whereas the apoptotic index (3.6 ± 0.6 vs 12.2 ± 1.7, p < 0.01) and the global level of H3K9ac and H3K27me3 were lower (p < 0.05) in the blastocysts produced from EC than in those produced from LC embryos. The relative transcript level of CASPASE3, CASPASE7, DNMT1, DNMT3a and CDX2 was higher (p < 0.05) and that of SOX2 was lower (p < 0.05) in blastocysts produced from LC than in those produced from EC embryos, whereas the expression level of CASPASE6, P53, P21, HDAC1, OCT4 and NANOG was not significantly different between the two groups. These results show that (i) following HMC, blastocysts produced from embryos that cleave early differ from those produced from late cleaving embryos in terms of epigenetic status and expression level of many important apoptosis-, pluripotency-, trophectoderm- and epigenetics-related genes, and (ii) EC embryos are superior to LC embryos in view of their higher developmental competence and quality.

The nuclear pore complex (NPC) is a highly modular assembly of 34 distinct nucleoporins (Nups), to form a versatile transport channel between the nucleus and cytoplasm. Among them, Nup62 is known as an essential component for nuclear transport while, Nup93 for the proper nuclear envelope assembly. These Nups constitute various NPC subcomplexes: such as central transport channel (CTC), cytoplasmic ring (CR) and inner ring (IR). However, how they play their role in the NPC assembly and transport activity is not clear. Here we delineated the interacting regions, conducted biochemical reconstitution and structural characterization of the mammalian CR complex to reveal its intrinsic dynamic behaviour and a distinct ‘4’ shaped architecture resembling the CTC complex. Our in vitro reconstitution data demonstrate that Nup62 coiled-coil domain is critical to form both Nup62322-525•Nup88517-742 and Nup62322-525•Nup88517-742•Nup214693-926 heterotrimers and both can bind to the Nup931-150. We therefore propose that the Nup93 act as a ‘sensor’ to bind to Nup62 shared heterotrimers including Nup62•Nup54 heterotrimer of the CTC, which was not shown previously as an interacting partner. Altogether, our biochemical study suggests that the Nup62 via its coiled-coil domain is central to form compositionally distinct yet structurally similar heterotrimers, and the Nup93 binds these diverse heterotrimers non-selectively.

The nuclear pore complex (NPC) is a highly modular assembly of 34 distinct nucleoporins (Nups), to form a versatile transport channel between the nucleus and cytoplasm. Among them, Nup62 is known as an essential component for nuclear transport while, Nup93 for the proper nuclear envelope assembly. These Nups constitute various NPC subcomplexes: such as central transport channel (CTC), cytoplasmic ring (CR) and inner ring (IR). However, how they play their role in the NPC assembly and transport activity is not clear. Here we delineated the interacting regions, conducted biochemical reconstitution and structural characterization of the mammalian CR complex to reveal its intrinsic dynamic behaviour and a distinct 4 shaped architecture resembling the CTC complex. Our data demonstrate that Nup62 coiled-coil domain is critical to forming both Nup62-Nup88 and Nup62-Nup88-Nup214 heterotrimers and both can bind to the Nup93. We therefore propose that Nup93 act as a sensor to bind to Nup62 shared heterotrimers including Nup62-Nup54 heterotrimer of the CTC, which was not shown previously as an interacting partner. Altogether, our study establishes that the Nup62 via its coiled-coil domain is central to form compositionally distinct yet structurally similar heterotrimers, and the Nup93 anchors these diverse heterotrimers by recognizing them non-selectively.