GANP (Germinal-center Associated Nuclear Protein) is a 210 kDa protein that is upregulated in germinal-centre B cells
27 and also in a variety of lymphomas.
28 GANP has been proposed to have a role in the immune response through generation of antigen-specific and high-affinity B cells during maturation in germinal-centres.
29 GANP has also been reported to suppress DNA recombination.
30 However, these observations do not explain why GANP is expressed in essentially all mammalian cells. Furthermore, because GANP also contains a region identical to MCM3AP (MCM3 acetylating protein), that interacts with and weakly acetylates MCM3,
31,32 these proteins have often been confused in databases and the literature. The nucleotide sequence for MCM3AP is completely contained in the 3′ region of GANP, suggesting that MCM3AP is a splice variant
33 and, furthermore, MCM3AP can be transcribed independently of GANP. Consequently, they should be referred to as independent, overlapping genes and not described interchangeably as at present in databases.
Recent work has identified GANP as an integral component of the mammalian bulk mRNA export machinery.
34 Separate domains of GANP show local homology to FG-nucleoporins, the yeast mRNA export factor Sac3p and the mammalian MCM3 acetyltransferase. GANP therefore combines features from different components of the mRNA export machinery and NPCs, and so has the potential to coordinate key steps in the gene expression pathway. In support of this hypothesis, GANP is localized predominantly to the nuclear face of the nuclear envelope, albeit combined with weaker staining of the nuclear interior.
34 Although it is not known precisely how GANP is targeted to the nuclear periphery, in yeast the CID motif (to which Sus1 and Cdc31 bind) targets Sac3 to the NPC.
20,35 The CID motif appears to be conserved in GANP,
20 suggesting it could have an analogous role for targeting GANP to NPCs. Also, the TREX-2 complex may be conserved in mammals, as Centrin-2, the human homolog of Cdc31, functions in mRNA export in vertebrates.
36GANP depletion inhibits mammalian mRNA export and causes nuclear accumulation of poly(A) + RNA with a concomitant decrease in levels in the cytoplasm.
34 Poly(A) + RNA does not accumulate at the nuclear envelope of GANP depleted cells, but instead accumulates in a distinct punctate focal pattern throughout the nucleus, excluding nucleoli. A similar pattern of poly(A) + RNA localization is observed in cells depleted of NXF1, the major mRNA export factor in mammalian cells
5,7 and resembles that seen for RNA polymerase II,
37 suggesting that GANP depletion results in an accumulation of nuclear mRNP complexes localized at, or near, sites of transcription. Moreover, GANP is associated with nuclear mRNPs and inhibiting transcription causes GANP to accumulate in foci throughout the nucleus, with a corresponding decrease in levels at NPCs.
34 Taken together, these findings suggest that GANP is mobile and that it shuttles between transcription factories and NPC's.
How might GANP facilitate efficient mRNA export in mammalian cells? NXF1 is considered to be the major exporter of bulk mRNA in metazoans
5,7 and has a similar depletion phenotype to GANP. Yeast Sac3, which is homologous to a local region of GANP, interacts in vitro with Mex67, the yeast homologue of NXF1, although the molecular basis of this interaction has not been established.
21 GANP interacts directly with NXF1 through its N-terminal fragment that contains FG motifs and overexpression of this fragment causes nuclear accumulation of both NXF1 and mRNA.
34GANP and NXF1 share several key features. Both associate with nuclear mRNPs, and both interact with NPCs. Furthermore, depletion of either protein from human cells results in retention of mRNPs in nuclear foci and not at NPCs.
34 However, NXF1 localizes predominantly in nuclear foci, with less staining at NPCs, whereas GANP localizes predominantly at NPC's, with a corresponding decrease in the nuclear interior.
34 Also, GANP interacts directly with the C-terminal domain of NXF1,
34 that interacts with FG-repeat nucleoporins.
2,3,6,38,39 Taken together, these results suggest that GANP may not fit the classical definition of an adaptor protein that recruits NXF1 to mRNPs. Adaptor proteins such as the SR proteins and ALY are localized predominantly in nuclear speckles and not at NPC's,
10,40 in contrast to GANP and NXF1. Furthermore, the SR proteins and ALY bind to the N-terminal domains of NXF1,
9,11 not the C-terminal NTF2-like and UBA-like domains that interact with FG-repeat nucleoporins and GANP. The in vivo localisation and interaction data favor GANP functioning in a later step in the export pathway, namely recruiting NXF1-containing mRNPs in the nuclear interior and delivering them to the NPC.
GANP contains a C-terminal domain that is identical to MCM3AP (MCM3 acetylating protein), that interacts with and acetylates MCM3, albeit weakly, or histones even more weakly.
31,32 As GANP contains the whole of MCM3AP within its sequence, it is possible that GANP might also possess acetyltransferase activity, though this has not been detected to date. It remains possible that acetylation might be a mechanism used during mRNA export to convert mRNPs from an export-inactive state to an export-active one or vice versa. Indeed, it has been shown that acetylation itself can result in functional chromatin reorganisation at mammalian nuclear pore complexes.
23 Furthermore, the SAGA transcription activation complex, of which histone acetyltransferase Gcn5 is a member, generates a number of histone modifications including acetylation.
41 Along with its role in transcription activation, SAGA is also required for mRNA export through its component Sus1/ENY2.
42,43 Therefore acetylation as a mechanism for regulating mRNA export deserves further examination.
1,2 Murray Stewart,2 and Ronald A Laskey1
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