November 1, 1992;
Nuclear transport and phosphorylation of the RNA binding Xenopus zinc finger protein XFG 5-1.
XFG 5-1 is a Krüppel-type Xenopus zinc finger protein with specific RNA homopolymer binding activity in vitro. In the oocyte
, the protein is distributed between nucleus
; the nuclear fraction, not the cytoplasm
, contains phosphorylated isoform(s) of XFG 5-1. In vitro phosphorylation by use of oocyte
/egg extracts or purified casein kinase II is specific to the amino-terminal portion of the protein. The carboxy-terminal zinc finger domain contains a signal sufficient for nuclear transport. Overexpression of either full length XFG 5-1 or of the carboxy-terminal portion alone, which maintains RNA binding and nuclear import activities, was achieved in Xenopus embryos by mRNA injection. This treatment did not result in impaired regulation of development, suggesting that XFG 5-1 functions in a way distinct from the mode of action exemplified in the Drosophila zinc finger protein Krüppel.
[+] show captions
Fig. 1. Purification and reactivity of antibodies directed against
XFG5-1. (A) Aliquots of bacterially expressed XFG 5-1/~-galactosidase
fusion proteins were subjected to Western-blot analysis with
antibodies before (lane 1) and after (lane 2) affinity purification (as
detailed in Materials and Methods). Protein signals corresponding to
the XFG 5-1/13-galactosidase fusion protein or to 13-galactosidase
are indicated. (B) XFG 5-1 protein was translated in vitro in the
presence of 3SS-methionine from in vitro transcribed RNA by use of
rabbit reticulocyte lysate. Protein samples were analyzed by SDS gel
electrophoresis either directly (lane 1) or after immunoprecipitation
with XFG 5-1 antibodies (lanes 2 and 3) and with control serum from
other hyperimmune rabbits (lanes 4 and 5); the molecular mass of
marker proteins (lane M) is indicated.
Fig. 2. XFG 5-1 protein expression in early Xenopus development
and in adult tissue. Oocyte, embryo and tissue extracts were subjected
to SDS gel electrophoresis, 80/~g of total protein were loaded
per lane (O, large oocyte; E, egg; B, blastula; G, gastrula; LG, late
gastrula; N, neurula; S, somite-stage; L, liver; T, testis) and subjected
to Western-blot analysis with either immunopurified XFG 5-1 antiserum
(upper portion) or antiserum against Xenopus enolase (bottom
portion). The positions of protein signals specific for XFG 5-1
and enolase are indicated.
Fig. 3. Nuclear protein preparations contain phosphorylated isoforms of XFG 5-1; Control oocytes and oocytes injected with XFG 5-1 mRNA
were manually dissected into nuclear and cytoplasmic fractions. Resulting protein preparations (8% SDS-PAGE) were subjected to Western-blot
analysis with immunopurified XFG 5-1 antibodies. Individual lanes contain five equivalents of total oocytes (T), cytoplasm (C) or nucleus (N).
The positions of marker proteins (M) and the signal specific for the phosphorylated form of XFG 5-1 are indicated. An additional set of nuclear
protein preparations from microinjected oocytes was treated with increasing concentrations of alkaline phosphatase (as indicated in U/ml) prior
to Western-blot analysis (10% SDS-PAGE).
Fig. 4. Phosphorylation of XFG 5-1 in vitro. (A) Bacterially expressed /3-galactosidase (0), or fusions with either full length XFG 5-1 (1), the
amino-terminal non-finger repeats (2) or the carboxy-terminal zinc finger domain (3) were immunoprecipitated and incubated with oocyte (O) or
egg (E) extracts in the presence of [y-32P]ATP. Phosphorylation was monitored by autoradiography after SDS gel electrophoresis. (B) Fusion
proteins (labelled as in A) were incubated with purified casein kinase II (CKII) and [y-3~P]ATP; protein phosphorylation was analyzed as in A.
(C) Coomassie stained SDS gel of the different protein preparations employed in A and B (as labelled in A).
Fig. 5. Nuclear import of XFG 5-1//3-galactosidase fusion proteins in Xenopus oocytes. Oocytes were injected with full-length XFG 5-1, the
carboxy-terminal portion (XFG 5-1 AN), and the amino-terminal portion (XFG 5-1 ,..4C), all fused to /3-galactosidase (I, input) Nucleoplasmin/
/3-galactosidase fusion protein was microinjected as a positive control for nuclear import, /3-galactosidase alone as a negative control. After
overnight incubation, oocytes were microdissected into nuclear (N) and cytoplasmic fractions (C); these protein fractions, as well as total oocyte
extract (T) were tested for the presence of microinjected proteins by immunoprecipitation followed by Western-blot analysis. Antibodies specific
for/3-galactosidase were utilized in both steps. The position and identity of individual fusion proteins is indicated; commercial /3-galactosidase
was used as a reference (M).
Fig. 6. Overexpression of XFG 5-1 protein in Xenopus embryos. (A)
Western-blot analysis of total protein preparations from fulMength
XFG 5-1 mRNA injected embryos. 2.5 embryo equivalents from
non-injected controls and from injected samples were applied per
lane after 6, 28 and 70 h of development. The two different protein
preparations from mRNA injected 28-h embryos were from samples
which had undergone normal (right hand lane) and abnormal (left
hand lane) gastrulation. The positions of reference molecular mass
markers (M) are indicated. (B) Immunoprecipitation of 3sS-labelled
proteins from oocytes injected with synthetic mRNA encoding the
carboxy-terminal fragment of XFG 5-1 (S) or from non-injected
control oocytes (C) with antibodies specific for XFG 5-1 before
affinity purification. The positions of XFG 5-1 ~IN (arrow) and of
reference molecular mass markers (M) are indicated.
Fig. 7. Development of XFG 5-1 mRNA injected Xenopus embryos
proceeding normally. Tadpoles were reared from either XFG 5-1
mRNA injected (A) or non-injected control (B) fertilized eggs. These
are representative examples of experiments summarized in Table 1.