We have annotated 20 predicted C. elegans
genes as encoding nucleoporins and begun their functional characterization. The number of nucleoporin genes is lower than found in mammals, where 28 genes have been described. It is possible that the nematode genome encodes fewer nucleoporins than higher eukaryotes. However, because yeast also contain ~30 different nucleoporins, we find this unlikely. There are, however, no additional nematode genes that exhibit nucleoporin-like sequence features. Electron microscopy of the NE in C. elegans
embryos has recently revealed that the NPC in this organism seems to have intermediate dimensions between vertebrate and yeast NPCs (Cohen et al., 2002
). We therefore suggest that the lack of additional nucleoporins in the database may be due to one or more of the following reasons. 1) Although the WormPep database has a high fidelity a certain number of genes are mispredicted (Reboul et al., 2001
) and perhaps existing genes are not yet included in the list of predicted ORFs. 2) Some C. elegans
nucleoporins might be so divergent from their mammalian counterparts that they will only be identified by direct functional analysis. 3) Eight of the 20 C. elegans
nucleoporin genes are predicted to encode two to three different isoforms, which could potentially increase the total number of nematode nucleoporin proteins without requirement for additional nucleoporin genes.
We have demonstrated that depletion of 12 of 20 individual nucleoporins causes almost complete embryonic lethality (CeNup35, -45/58, -54, -62, -93, -98/96, -153, -155, -160, -205, -358, and CeSec13R). This is in agreement with yeast data showing that ~50% of nucleoporin genes are essential for viability (Fabre and Hurt, 1997
). Combinatorial RNAi revealed that a further five npp genes are important for early nematode development (CeNup85, -107, -133, -214, and CeRAE1). Only three npp genes did not reveal any phenotype when targeted alone or in combination (CeNup50, CeSeh1, and Cegp210). Based on our results and those of others, we speculate that CeNup50 and CeSeh1 might be dispensable for embryonic development, whereas Cegp210 may have escaped depletion by RNAi. Reverse transcription-PCR analysis of mRNA from CeNup50(RNAi) and CeSeh1(RNAi) embryos show a specific although limited reduction in the amount of targeted mRNA, whereas we did not detect any significant decrease of Cegp210 mRNA in Cegp210(RNAi) embryos (our unpublished data). Other laboratories have reported Cegp210(RNAi) phenotypes ranging from wild-type (Fraser et al., 2000
; Piano et al., 2002
) to weak embryonic lethality (Maeda et al., 2001
; Colaiacovo et al., 2002
; Cohen et al., 2003
), probably reflecting differences in RNAi efficiency. Cegp210 is the only transmembrane nucleoporin recognizable from its sequence and it might therefore be expected to be important for NPC anchoring, as indeed suggested by recent work from Gruenbaum and colleagues (Cohen et al., 2003
When analyzed by DIC microscopy the RNAi phenotypes came in two categories. When CeNup54, -45/58, -62, -85, -93, or -205 were targeted by RNAi, the nuclei of early embryos were slightly smaller than wild type but seemed otherwise normal. However, because the majority [≥87% except for CeNup85(RNAi)] of these embryos died before hatching, these particular nucleoporins are essential for development. A more striking phenotype was observed when another set of nucleoporins was depleted. RNAi depletion of CeNup35, -98/96, -153, -155, -160, -358, or CeSec13 seemed to completely block nuclear formation after the first mitosis. This indicates that certain nucleoporins in addition to their expected roles in nucleocytoplasmic transport are required for nuclear reassembly after mitosis. We have previously analyzed the defects in nuclear envelope formation upon depletion of CeNup358 (Askjaer et al., 2002
) and are currently investigating the remaining nucleoporins with this particular phenotype. It should be noted that for six nucleoporin genes DIC phenotypes similar to those presented here have been reported previously (CeNup98/96 and -358, Gönczy et al., 2000
; CeNup62, -85, -153, and -160, Zipperlen et al., 2001
). Our description of the remaining seven npp genes with identifiable early DIC phenotypes demonstrates that a large number of nucleoporins are required for postmitotic nuclear reassembly and function. In particular, our analysis provides a first demonstration of a nuclear-related function of the novel nucleoporin CeNup35 (Cronshaw et al., 2002
). Finally, the observation that combinatorial RNAi against CeNup85, -107, and -133 causes dramatic nuclear morphology defects suggests that these nucleoporins, which are known to physically interact in other organisms, are individually dispensable but are together essential for nuclear assembly similar to the situation with components of the analogous yeast Nup84p complex (Fabre and Hurt, 1997
The lethality associated with essential nucleoporin genes that are not required for nuclear formation in early embryos could be caused by defects in nucleocytoplasmic transport. Imbalance in concentrations of macromolecules across the nuclear envelope would then in turn presumably have consequences on e.g., chromatin maintenance or gene expression patterns. Our analysis of CeNup205 and CeNup93 has demonstrated that chromosome behavior inside the nucleus is influenced by these nucleoporins. In all embryonic nuclei the chromosomes displayed a strong and abnormal peripheral condensation upon depletion of CeNup205 or CeNup93. In contrast, nuclear protein import was not blocked in these embryos, as visualized by nuclear accumulation of YFP-lamin, indicating that the changes in chromatin morphology were probably not caused by a lack of protein import. These observations extend and provide important support for previous in vitro data. Nup93 interacts with Nup188 and Nup205 in Xenopus
egg extracts (Grandi et al., 1997
; Miller et al., 2000
). Depletion of Nup93 from such extracts leaves nuclear protein import unaffected while causing a delay in DNA replication (Grandi et al., 1997
), which might be related to the perinuclear chromatin condensation defect observed here.
However, we also observed significant differences in the phenotypes caused by depletion of these nucleoporins compared with previous data. Depletion of CeNup205 or CeNup93 led to strong aggregation of NPCs in the NE, as judged by NPC staining by mAb mAb414. The total mAb414 signal was however not noticeably decreased, indicating that the NEs in affected embryos contained roughly the same number of NPCs but in a clustered distribution. In contrast, nuclei assembled in vitro in Xenopus
extracts immunodepleted for Nup93 react much less with mAb414 (Grandi et al., 1997
). Due to the stability of NPC subcomplexes, depletion by affinity purification in general leads to codepletion of several components concomitantly, thereby obscuring the assignment of specific functions to individual nucleoporins. We therefore propose that the more dramatic effects seen on the number of NPCs upon immunodepletion of Nup93 compared with our RNAi depletion of CeNup93 may have resulted from codepletion of other factors with Nup93 from the in vitro assembly reactions. RNAi is in general considered as an efficient tool to deplete single proteins in vivo. However, depletion of nucleoporins in mammalian tissue culture cells by RNAi has recently revealed an unexpected cross-interference with the accumulation of other nucleoporins, presumably at the posttranslational level (Boehmer et al., 2003
; Harel et al., 2003
; Walther et al., 2003
). To address this, we analyzed the protein concentrations of four different nucleoporins in embryos depleted of either of six essential nucleoporins. In none of these experiments did we observe cross-interference, which leads us to conclude that our observations can most likely be attributed to depletion of individual nucleoporins.
In the absence of CeNup205 or CeNup93 a dramatic failure in the nuclear exclusion of ~70-kDa fluorescent reporter molecules (GFP-β-tubulin and fluorescent dextrans) was observed. However, a larger dextran of ~160 kDa was excluded from the nuclei of both depleted and control embryos, indicating that the nuclei were enclosed by intact NEs. This was further supported by the finding that the inner NE proteins MAN1 and emerin were present around nuclei of CeNup205(RNAi) and CeNup93(RNAi) or doubly depleted embryos. In combination, these data demonstrated that CeNup205 are CeNup93 are involved in determination of the size exclusion limit of the NPC permeability barrier, possibly by forming part of the central channel in the NPC. In S. cerevisiae
, Nup188p has previously been implicated in control of nuclear pore resting size (Shulga et al., 2000
). Although we were unable to identify a C. elegans
homolog of Nup188p, it is intriguing that Nup188p, which itself is nonessential, interacts with the two yeast homologs of CeNup205 and CeNup93, Nup192p and Nic96p, respectively.
Conditional mutations in the S. cerevisiae
cause defects in NPC assembly leading to a lower number of NPCs in the NE (Kosova et al., 1999
; Gomez-Ospina et al., 2000
). This might seem contradictory to our results. However, neither of the two mutations are null alleles (null mutants of NUP192
are nonviable), which means that mutant protein is produced in these strains. A likely possibility, based on the published data and our results, is that the mutant yeast proteins are unable to be integrated into the NE or NPCs but can still interact with at least some of their normal interaction partners, thereby sequestering nucleoporins from the NE. In contrast, in RNAi-depleted embryos the absence of CeNup205 or CeNup93 does not prevent NPC biogenesis but the NPCs formed have an increased resting pore size.
The fact that perinuclear chromatin condensation and lack of proper nuclear exclusion were both already observed in two-cell embryos suggest a connection between the two processes. Chromosome condensation needs to be precisely regulated to allow proper access of transcription and replication factors to the DNA in interphase and S phase and efficient chromosome alignment and segregation during mitosis (Jessberger, 2002
). In vertebrates, the five-subunit condensin complex is responsible for chromosome condensation and condensation is at least partially controlled by the nucleocytoplasmic distribution of condensin (Schmiesing et al., 2000
). In the light of the observations presented here, it is possible that the increase in permeability seen in CeNup205(RNAi) and CeNup93(RNAi) embryos allows premature entry of condensin-like factors.
In summary, our study demonstrates that the C. elegans
genome encodes orthologs of many of the nucleoporins found in vertebrates. More than 50% of these proteins are required for embryonic development. Detailed functional analysis of CeNup205 and CeNup93, two components of a conserved NPC subcomplex, demonstrates that they are important structural components of the NPC that participate in the establishment of the NPC resting pore size. The NPC diffusion limit is controlled during the cell cycle (Feldherr and Akin, 1990
) and increases as a very early step in nuclear envelope breakdown in starfish oocytes (Lenart et al., 2003
). Interestingly, this latter increase in pore size was correlated with partial NPC disassembly. Further work is required to analyze whether Nup205 and Nup93 have a role in this process.