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Identification, developmental expression and regulation of the Xenopus ortholog of human FANCG/XRCC9.
Stone S
,
Sobeck A
,
van Kogelenberg M
,
de Graaf B
,
Joenje H
,
Christian J
,
Hoatlin ME
.
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Fanconi anemia (FA) is associated with variable developmental abnormalities, bone marrow failure and cancer susceptibility. FANCG/XRCC9 is member of the FA core complex, a group of proteins that control the monoubiquitylation of FANCD2, an event that plays a critical role in maintaining genomic stability. Here we report the identification of the Xenopus laevis ortholog of human FANCG (xFANCG), its expression during development, and its molecular interactions with a partner protein, xFANCA. The xFANCG protein sequence is 47% similar to its human ortholog, with highest conservation in the two putative N-terminal leucine zippers and the tetratricopeptide repeat (TPR) motifs. xFANCG is maternally and zygotically transcribed. Prior to the midblastula stage, a single xFANCG transcript is observed but two additional alternatively spliced mRNAs are detected after the midblastula transition. One of the variants is predicted to encode a novel isoform of xFANCG lacking exon 2. The mutual association between FANCG and FANCA required for their nuclear import is conserved in Xenopus egg extracts. Our data demonstrate that interactions between FANCA and FANCG occur at the earliest stage of vertebrate development and raise the possibility that functionally different isoforms of xFANCG may play a role in early development.
Figure 1 Comparative sequence analysis of Xenopus and human FANCG. A ClustalW alignment of full-length Xenopus laevis FANCG
(xFANCG) with full-length human FANCG (hFANCG). Dark grey shading indicates amino acid identity; light grey shading indicates
amino acid similarity. The exons described in this paper are outlined with black boxes—labeled (on top) 1, 2 and 3. Asterisks indicate
positions of serines shown to be important for hFANCG function (Mi et al. 2004; Qiao et al. 2004). Black arrows indicate the critical
leucine residues of the previously described leucine zipper motifs (Demuth et al. 2000). Grey boxes indicate TPR motifs—labeled (on
bottom) 1–7 (Blom et al. 2002). The black underlined sequence (amino acids 378–407) shares homology with the transcriptional
co-repressor NAB1(van De Vrugt et al. 2002).
Figure 2 An mRNA splice variant of Xenopus FANCG, lacking exon 2 is present during embryo development. (A) A ClustalW
alignment shows the nucleotide sequence of hFANCG cDNA compared with full length xFANCG and an xFANCG deletion variant
that lacks exon 2 (xFANCGshort). The borders of exons 1, 2 and 3 are conserved between human and Xenopus as indicated by black arrows.
(B) A schematic of the alternative splice event that leads to skipping of exon 2 in xFANCGshort. Line 1: 5′ cDNA sequence of full-length
xFANCG encompassing exons 1–3. Line 2: Exon–intron structure of xFANCG on the genomic level. Introns are indicated as black lines.
The boxed region represents the start codon (ATG) for full-length xFANCG. Line 3: cDNA sequence of the alternative splice variant
xFANCGshort, which is missing exon 2. A putative start codon is indicated by a boxed ATG located at the end of exon 1 which is in
frame with exon 3 (+2 reading frame) but not with the original start codon at the beginning of exon 1. (C) A ClustalW alignment shows
the translation product of exons 1–3 of hFANCG compared with that of full-length xFANCG and xFANCGshort. Black arrows indicate
the borders of exons 1, 2 and 3.
Figure 3 xFA genes are expressed maternally and during
embryonic development. (A) An agarose gel shows the RT-PCR
products for xFANCG -A, -D2, -F and -L from different
developmental stages (as indicated at the top of panel 1) using the
primers listed in Materials and methods. Transcripts of all FA genes
were expressed throughout development (panels 1–5). Three
alternative transcripts were detected for xFANCG at stages 20, 24,
33, 39 and 41 (after the midblastula transition at stage 8, panel 1).
(B) An immunoblot probed with anti-xFANCG, anti-xFANCA,
and xFANCD2 indicated on the left of each panel, shows that
xFANCG, xFANCA and xFANCD2 are expressed throughout
the developmental stages indicated (top). The right-most lane is a
negative control (labeled “Pre”) consisting of a re-probe of the same
immunoblots with the pre-immune antisera corresponding to the
antibody listed at the left of each panel.
Figure 4 Interactions between human FANCA and FANCG are conserved in Xenopus egg extracts. (A) An immunoblot (antibodies
used are indicated on left of each panel) shows that an immunoprecipitation (IP) with anti-xFANCG (lane 3, panel 2), coimmunoprecipitated
(co-IPed) xFANCA (lane 3, panel 1) but not xFANCD2 (lane 3, panel 3). An IP with anti-xFANCA (lane 4, panel 1)
co-IPed xFANCG (lane 4, panel 2) but not xFANCD2 (lane 4, panel 3). An IP with anti-xFANCD2 (lane 2, panel 3) did not co-IP
xFANCA (lane 2, panel 1) or xFANCG (lane 2, panel 2). One microliter of egg extract was used as a positive control for all three proteins
(lane 1, all panels). Lane 5 is a mock control IP with xFANCG pre-immune sera. (B) An immunoblot (antibodies used are indicated on
left of each panel) shows the protein complex elution profile of egg extract from a Superose 6 gel filtration column. The majority of
xFANCG and xFANCA proteins co-eluted similarly at ∼900 kDa. (C) An immunoblot (antibodies used are indicated on left of each
panel) shows that in Xenopus extracts (lanes 1–3) depletion of xFANCA (lane 1, panel 2) partially co-depleted xFANCG (lane 1, panel 3)
and a depletion of xFANCG (lane 2, panel 3) partially co-depleted xFANCA (lane 2, panel 2) when compared to an IgG control
depletion (lane 3, panels 2 and 3). The bands indicated as xFANCG have been identified by mass spectrometry (S. Stone, unpublished
data). xFANCD2 was used as a loading control for the first three lanes (panel 1). After the formation of nuclei in the depleted extracts
(lanes 4–6), there was no nuclear import of residual non-depleted xFANCA or xFANCG (lanes 4 and 5, panels 2 and 3) following the
depletion of either protein, whereas the IgG control depleted nuclei shows normal import of xFANCA and xFANCG (lane 6, panels 2
and 3). Although imported into the nuclei (panel 1, lanes 4–6), xFANCD2 was not monoubiquitylated in the absence of xFANCA or
xFANCG (lanes 4 and 5, panel 1) but was monoubiquitylated in the IgG control depleted nuclei (lane 6, panel 1).