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

Summary 2
We and others have shown that human NXF proteins function as heterodimers
bound to the small protein p15. Accordingly, silencing of Drosophila p15 resulted in a
block of mRNA export which was indistinguishable from the export inhibition seen after
targeting NXF1. These observations indicated that neither NXF1 nor p15 can promote
export in the absence of the other subunit of the heterodimer.
NXF1:p15 heterodimers are implicated in late steps of mRNA export, i.e. in the
translocation of mRNP export cargoes across the nuclear pore complex. The mechanism by
which NXF1:p15 dimers are recruited to the mRNA is unclear. A protein that is thought to
play a role in this process is the putative RNA helicase UAP56. Similar to NXF1 and p15,
UAP56 was shown to be essential for mRNA export in Drosophila. UAP56 is recruited
cotranscriptionally to nascent transcripts and was suggested to facilitate the interaction of
NXF1:p15 with mRNPs.
Even though both NXF1:p15 heterodimers and UAP56 had been implicated in
general mRNA export, it was unclear whether there are classes of mRNAs that require
NXF1:p15, but not UAP56 or vice versa. It was also unclear what fraction of cellular
mRNAs is exported by NXF1:p15 dimers and UAP56, and whether mRNAs exist that
reach the cytoplasm through alternative routes, i.e. by recruiting other export receptors.
To address these issues we performed a genome-wide analysis of nuclear mRNA
export pathways using microarray technology (2.2.2). We analyzed the relative abundance
of nearly half of the Drosophila transcriptome in the cytoplasm of Drosophila Schneider
cells depleted of different export factors by RNAi. We showed that the vast majority of
transcripts were underrepresented in the cytoplasm of cells depleted of NXF1, p15 or
UAP56 as compared to control cells. Only a small number of mRNAs were apparently not
affected by the depletions. These observations, together with the wide and similar effects
on mRNA levels caused by the depletion of NXF1, p15 or UAP56, indicate that these
proteins define the major mRNA export pathway in these cells. We also identified a small
subset of mRNAs which appeared to be exported by NXF1:p15 dimers independently of
UAP56.
In contrast, no significant changes in mRNA expression profiles were observed in
cells depleted of NXF2 or NXF3, suggesting that neither NXF2 nor NXF3 play an
essential role in mRNA export in Drosophila Schneider cells.
Crm1 is a transport receptor implicated in the export of a variety of non-mRNA
and protein cargoes. In addition, human Crm1 has been suggested to be involved in the
export of a specific mRNA species, serving as a "specialized" mRNA export receptor. A
role of human Crm1 in the export of bulk mRNA is considered unlikely. We analyzed the
role of Drosophila Crm1 in mRNA export by inhibiting Crm1 with the drug leptomycin B
in Schneider cells. Subsequent microarray analysis demonstrated that the inactivation of