The role of human and Drosophila NXF proteins in nuclear mRNA ...

2.1 The nuclear export factor family:
characterization of human NXF proteins
Results/Publications 34
2.1.1 Paper 1
TAP (NXF1) belongs to a multigene family of putative RNA export factors
with a conserved modular architecture
Herold, A., Suyama, M., Rodrigues, J. P., Braun, I., Kutay, U., Carmo-Fonseca,
M., Bork, P., and Izaurralde, E. (2000) Mol Cell Biol 20, 8996-9008.
Context
The human protein TAP and its yeast orthologue Mex67p have both been
implicated in the nuclear export of mRNAs. Both TAP and Mex67p act as heterodimers
together with a small cofactor called p15 in humans and Mtr2p in yeast. The fact that
overexpression of TAP and p15 in S. cerevisiae partially restores growth of the otherwise
lethal mex67/mtr2 double knockout, suggests that the mRNA export pathway mediated by
TAP/Mex67p has been conserved throughout eukaryotic evolution (Katahira et al., 1999).
As both proteins show significant homology and share a similar domain organization
(Segref et al., 1997), we hypothesized that if other proteins with similarity to TAP existed,
they might also play a role in mRNA export. To identify such putative mRNA export
factors, we performed an extensive search for TAP and p15 homologues in higher
eukaryotes and combined this with a functional characterization of selected human TAP
and p15 homologues.
Summary of results and conclusions
In this study we identified two TAP-related sequences in the C. elegans genome,
four in the Drosophila genome and, in addition to TAP itself, five in the human genome
(of which one might be, and a second one certainly is a pseudogene). This conserved
family of TAP homologues was termed NXF (nuclear export factor) family. We showed
that the overall domain organization and residues critical for p15 or nucleoporin binding
are conserved in most predicted family members. Two human homologues (NXF2 and
NXF3) were cloned and characterized in more detail. We demonstrated that, like TAP,
NXF2 interacts with the RNA-binding proteins E1B-AP5 and REF1-II, as well as with
RNA in vitro, while NXF3 binds only to E1B-AP5. In contrast to TAP, neither NXF2 nor
NXF3 interacts with the CTE of simian retrovirus type 1 in vitro. Accordingly neither of
them promotes the nuclear export of a CTE-containing reporter RNA in quail cells. NXF2
and NXF3 were also tested for their capability to bind nucleoporins in vitro using pulldown
assays. In agreement with the truncation of the predicted nucleoporin-binding
domain in NXF3, only NXF2 was found to interact efficiently with CAN. NXF2 was also