Fig. 4. XRnf12 causes ubiquitination and proteasome-dependent degradation of Ldb1
in a RING-dependent manner. (A) XRnf12 does not affect the steady-state level
of Xlim-1-FLAG, either in the presence or absence of Ldb1. The indicated mRNAs
were injected into the ventral region and the expression levels of the
FLAG-tagged proteins were examined at the gastrula stage. Note that the levels
of Xlim-1-FLAG are increased in the presence of Ldb1. ß-tubulin, loading
control. Amounts of mRNAs injected (ng/embryo): Xlim-1-FLAG, 0.25; Ldb1, 0.5;
XRnf12, 0.25. (B) XRnf12 decreases the steady-state level of FLAG-Ldb1 both in
the presence and absence of Xlim-1 in a RING-dependent manner. Note the
increase in the expression level of FLAG-Ldb1 by Xlim-1 co-expression. Amounts
of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; Xlim-1, 0.25; XRnf12 constructs, 0.25.
(C) XRnf12 enhances ubiquitination of Ldb1. Embryos were injected ventrally
with the mRNAs indicated and the cell lysates were immunoprecipitated (IP)
with anti-FLAG antibody followed by either anti-FLAG or anti-HA immunoblotting
(IB) to detect non-ubiquitinated Ldb1 or ubiquitinated proteins, respectively.
Co-expression of XRnf12 results in downregulation of non-ubiquitinated
FLAG-Ldb1 expression levels (lower panel). While weak ubiquitination is
observed in the absence of XRnf12 (lane 4), strong ladder-like ubiquitination
signals appear in the presence of XRnf12 (lane 7). XRnf12(HC>AA) does not
enhance ubiquitination (lane 8). Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 2.0;
HA-Ub, 1.0; XRnf12 constructs, 1.0. (D) The N-terminal region (aa 1-291) of
Ldb1 is sufficient for ubiquitination by XRnf12. By using FLAG-Ldb1
C
instead of FLAG-Ldb1, smaller-sized ubiquitinated protein bands are detected,
confirming that the ubiquitinated proteins in C are indeed Ldb1 and not some
other proteins associated with Ldb1. Co-expression of XRnf12 also results in
downregulation of non-ubiquitinated FLAG-Ldb1C. The amounts of mRNAs
used are the same as in C. Arrowhead indicates the position of IgG. (E) XRnf12
causes proteasome-dependent degradation of Ldb1. After mRNA injection, cells
were dispersed and cultured in the presence or absence of MG-132 until the
gastrula stage. Decrease of FLAG-Ldb1 levels by XRnf12 (lane 5) is suppressed
in the presence of MG-132 (lane 6). MG-132 does not affect the expression of
FLAG-Ldb1 (lanes 3,4). Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; Xlim-1,
0.25; XRnf12, 0.25. (F) The steady-state level of FLAG-Ldb1 is downregulated
by hRNF6 and, to a lesser extent, by hRNF38, but not by hRNF13. The
experimental design is the same as in A and B. ß-tubulin, loading
control. Amounts of mRNAs (ng/embryo): FLAG-Ldb1, 0.5; RING finger proteins,
0.5. (G) RING finger proteins that cause reduction in the steady-state level
of Ldb1 interact with Ldb1. GST pull-down assay was performed with
35S-labeled XRnf12, hRNF6, hRNF13 and hRNF38. Human RNF13 does not
interact with GST-Ldb1, while other RING finger proteins do. GST serves as a
negative control.