|
Crystal B-evidence |
|
Structure I-evidence |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
Domain O |
|
of O |
|
the O |
|
Arabidopsis B-taxonomy_domain |
|
Flowering B-protein_type |
|
Regulator I-protein_type |
|
FPA B-protein |
|
|
|
The O |
|
Arabidopsis B-taxonomy_domain |
|
protein O |
|
FPA B-protein |
|
controls O |
|
flowering O |
|
time O |
|
by O |
|
regulating O |
|
the O |
|
alternative O |
|
3 O |
|
′- O |
|
end O |
|
processing O |
|
of O |
|
the O |
|
FLOWERING B-gene |
|
LOCUS I-gene |
|
( O |
|
FLC B-gene |
|
) O |
|
antisense B-chemical |
|
RNA I-chemical |
|
. O |
|
|
|
FPA B-protein |
|
belongs O |
|
to O |
|
the O |
|
split B-protein_type |
|
ends I-protein_type |
|
( O |
|
SPEN B-protein_type |
|
) O |
|
family O |
|
of O |
|
proteins O |
|
, O |
|
which O |
|
contain O |
|
N O |
|
- O |
|
terminal O |
|
RNA B-structure_element |
|
recognition I-structure_element |
|
motifs I-structure_element |
|
( O |
|
RRMs B-structure_element |
|
) O |
|
and O |
|
a O |
|
SPEN B-structure_element |
|
paralog I-structure_element |
|
and I-structure_element |
|
ortholog I-structure_element |
|
C I-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
( O |
|
SPOC B-structure_element |
|
) O |
|
domain O |
|
. O |
|
|
|
The O |
|
SPOC B-structure_element |
|
domain O |
|
is O |
|
highly B-protein_state |
|
conserved I-protein_state |
|
among O |
|
FPA B-protein |
|
homologs O |
|
in O |
|
plants B-taxonomy_domain |
|
, O |
|
but O |
|
the O |
|
conservation O |
|
with O |
|
the O |
|
domain O |
|
in O |
|
other O |
|
SPEN B-protein_type |
|
proteins O |
|
is O |
|
much O |
|
lower O |
|
. O |
|
|
|
We O |
|
have O |
|
determined O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
Arabidopsis B-species |
|
thaliana I-species |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
at O |
|
2 O |
|
. O |
|
7 O |
|
Å O |
|
resolution O |
|
. O |
|
|
|
The O |
|
overall O |
|
structure B-evidence |
|
is O |
|
similar O |
|
to O |
|
that O |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
in O |
|
human B-species |
|
SMRT B-protein |
|
/ I-protein |
|
HDAC1 I-protein |
|
Associated I-protein |
|
Repressor I-protein |
|
Protein I-protein |
|
( O |
|
SHARP B-protein |
|
), O |
|
although O |
|
there O |
|
are O |
|
also O |
|
substantial O |
|
conformational O |
|
differences O |
|
between O |
|
them O |
|
. O |
|
|
|
Structural B-experimental_method |
|
and I-experimental_method |
|
sequence I-experimental_method |
|
analyses I-experimental_method |
|
identify O |
|
a O |
|
surface B-site |
|
patch I-site |
|
that O |
|
is O |
|
conserved B-protein_state |
|
among O |
|
plant B-taxonomy_domain |
|
FPA B-protein |
|
homologs O |
|
. O |
|
|
|
Mutations B-experimental_method |
|
of O |
|
two O |
|
residues O |
|
in O |
|
this O |
|
surface B-site |
|
patch I-site |
|
did O |
|
not O |
|
disrupt O |
|
FPA B-protein |
|
functions O |
|
, O |
|
suggesting O |
|
that O |
|
either O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
is O |
|
not O |
|
required O |
|
for O |
|
the O |
|
role O |
|
of O |
|
FPA B-protein |
|
in O |
|
regulating O |
|
RNA B-chemical |
|
3 O |
|
′- O |
|
end O |
|
formation O |
|
or O |
|
the O |
|
functions O |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
cannot O |
|
be O |
|
disrupted O |
|
by O |
|
the O |
|
combination O |
|
of O |
|
mutations O |
|
, O |
|
in O |
|
contrast O |
|
to O |
|
observations O |
|
with O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
. O |
|
|
|
Eukaryotic B-taxonomy_domain |
|
messenger B-chemical |
|
RNAs I-chemical |
|
( O |
|
mRNAs B-chemical |
|
) O |
|
are O |
|
made O |
|
as O |
|
precursors O |
|
through O |
|
transcription O |
|
by O |
|
RNA B-complex_assembly |
|
polymerase I-complex_assembly |
|
II I-complex_assembly |
|
( O |
|
Pol B-complex_assembly |
|
II I-complex_assembly |
|
), O |
|
and O |
|
these O |
|
primary O |
|
transcripts O |
|
undergo O |
|
extensive O |
|
processing O |
|
, O |
|
including O |
|
3 O |
|
′- O |
|
end O |
|
cleavage O |
|
and O |
|
polyadenylation O |
|
. O |
|
|
|
In O |
|
addition O |
|
, O |
|
alternative O |
|
3 O |
|
′- O |
|
end O |
|
cleavage O |
|
and O |
|
polyadenylation O |
|
is O |
|
an O |
|
essential O |
|
and O |
|
ubiquitous O |
|
process O |
|
in O |
|
eukaryotes B-taxonomy_domain |
|
. O |
|
|
|
Recently O |
|
, O |
|
the O |
|
split B-protein_type |
|
ends I-protein_type |
|
( O |
|
SPEN B-protein_type |
|
) O |
|
family O |
|
of O |
|
proteins O |
|
was O |
|
identified O |
|
as O |
|
RNA B-protein_type |
|
binding I-protein_type |
|
proteins I-protein_type |
|
that O |
|
regulate O |
|
alternative O |
|
3 O |
|
′- O |
|
end O |
|
cleavage O |
|
and O |
|
polyadenylation O |
|
. O |
|
|
|
They O |
|
are O |
|
characterized O |
|
by O |
|
possessing O |
|
N O |
|
- O |
|
terminal O |
|
RNA B-structure_element |
|
recognition I-structure_element |
|
motifs I-structure_element |
|
( O |
|
RRMs B-structure_element |
|
) O |
|
and O |
|
a O |
|
conserved B-protein_state |
|
SPEN B-structure_element |
|
paralog I-structure_element |
|
and I-structure_element |
|
ortholog I-structure_element |
|
C I-structure_element |
|
- I-structure_element |
|
terminal I-structure_element |
|
( O |
|
SPOC B-structure_element |
|
) O |
|
domain O |
|
( O |
|
Fig O |
|
1A O |
|
). O |
|
|
|
The O |
|
SPOC B-structure_element |
|
domain O |
|
is O |
|
believed O |
|
to O |
|
mediate O |
|
protein O |
|
- O |
|
protein O |
|
interactions O |
|
and O |
|
has O |
|
diverse O |
|
functions O |
|
among O |
|
SPEN B-protein_type |
|
family O |
|
proteins O |
|
, O |
|
but O |
|
the O |
|
molecular O |
|
mechanism O |
|
of O |
|
these O |
|
functions O |
|
is O |
|
not O |
|
well O |
|
understood O |
|
. O |
|
|
|
Sequence B-evidence |
|
conservation I-evidence |
|
of O |
|
SPOC B-structure_element |
|
domains O |
|
. O |
|
|
|
Domain O |
|
organization O |
|
of O |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
FPA B-protein |
|
. O |
|
( O |
|
B O |
|
). O |
|
|
|
Sequence B-experimental_method |
|
alignment I-experimental_method |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
domains O |
|
of O |
|
Arabidopsis B-species |
|
thaliana I-species |
|
FPA B-protein |
|
, O |
|
human B-species |
|
RBM15 B-protein |
|
, O |
|
Drosophila B-taxonomy_domain |
|
SPEN B-protein_type |
|
, O |
|
mouse B-taxonomy_domain |
|
MINT B-protein |
|
, O |
|
and O |
|
human B-species |
|
SHARP B-protein |
|
. O |
|
|
|
Residues O |
|
in O |
|
surface B-site |
|
patch I-site |
|
1 I-site |
|
are O |
|
indicated O |
|
with O |
|
the O |
|
orange O |
|
dots O |
|
, O |
|
and O |
|
those O |
|
in O |
|
surface B-site |
|
patch I-site |
|
2 I-site |
|
with O |
|
the O |
|
green O |
|
dots O |
|
. O |
|
|
|
The O |
|
secondary O |
|
structure O |
|
elements O |
|
in O |
|
the O |
|
structure B-evidence |
|
of O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
are O |
|
labeled O |
|
. O |
|
|
|
Residues O |
|
that O |
|
are O |
|
strictly B-protein_state |
|
conserved I-protein_state |
|
among O |
|
the O |
|
five O |
|
proteins O |
|
are O |
|
shown O |
|
in O |
|
white O |
|
with O |
|
a O |
|
red O |
|
background O |
|
, O |
|
and O |
|
those O |
|
that O |
|
are O |
|
mostly B-protein_state |
|
conserved I-protein_state |
|
in O |
|
red O |
|
. O |
|
|
|
FPA B-protein |
|
, O |
|
a O |
|
SPEN B-protein_type |
|
family O |
|
protein O |
|
in O |
|
Arabidopsis B-species |
|
thaliana I-species |
|
and O |
|
other O |
|
plants B-taxonomy_domain |
|
, O |
|
was O |
|
found O |
|
to O |
|
regulate O |
|
the O |
|
3 O |
|
′- O |
|
end O |
|
alternative O |
|
cleavage O |
|
and O |
|
polyadenylation O |
|
of O |
|
the O |
|
antisense B-chemical |
|
RNAs I-chemical |
|
of O |
|
FLOWERING B-gene |
|
LOCUS I-gene |
|
( O |
|
FLC B-gene |
|
), O |
|
a O |
|
flowering O |
|
repressor O |
|
gene O |
|
. O |
|
|
|
FPA B-protein |
|
promotes O |
|
the O |
|
3 O |
|
′- O |
|
end O |
|
processing O |
|
of O |
|
class O |
|
I O |
|
FLC B-gene |
|
antisense B-chemical |
|
RNAs I-chemical |
|
, O |
|
which O |
|
includes O |
|
the O |
|
proximal O |
|
polyadenylation B-site |
|
site I-site |
|
. O |
|
|
|
This O |
|
is O |
|
associated O |
|
with O |
|
histone B-protein_type |
|
demethylase I-protein_type |
|
activity O |
|
and O |
|
down O |
|
- O |
|
regulation O |
|
of O |
|
FLC B-gene |
|
transcription O |
|
. O |
|
|
|
Although O |
|
a O |
|
SPOC B-structure_element |
|
domain O |
|
is O |
|
found O |
|
in O |
|
all O |
|
the O |
|
SPEN B-protein_type |
|
family O |
|
proteins O |
|
, O |
|
its O |
|
sequence O |
|
conservation O |
|
is O |
|
rather O |
|
low O |
|
. O |
|
|
|
For O |
|
example O |
|
, O |
|
the O |
|
sequence O |
|
identity O |
|
between O |
|
the O |
|
SPOC B-structure_element |
|
domains O |
|
of O |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
FPA B-protein |
|
and O |
|
human B-species |
|
SMRT B-protein |
|
/ I-protein |
|
HDAC1 I-protein |
|
Associated I-protein |
|
Repressor I-protein |
|
Protein I-protein |
|
( O |
|
SHARP B-protein |
|
) O |
|
is O |
|
only O |
|
19 O |
|
% O |
|
( O |
|
Fig O |
|
1B O |
|
). O |
|
|
|
Currently O |
|
, O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
is O |
|
the O |
|
only O |
|
one O |
|
with O |
|
structural O |
|
information O |
|
. O |
|
|
|
As O |
|
a O |
|
first O |
|
step O |
|
toward O |
|
understanding O |
|
the O |
|
molecular O |
|
basis O |
|
for O |
|
the O |
|
regulation O |
|
of O |
|
alternative O |
|
3 O |
|
′- O |
|
end O |
|
processing O |
|
and O |
|
flowering O |
|
by O |
|
FPA B-protein |
|
, O |
|
we O |
|
have O |
|
determined O |
|
the O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
of O |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
FPA B-protein |
|
at O |
|
2 O |
|
. O |
|
7 O |
|
Å O |
|
resolution O |
|
. O |
|
|
|
The O |
|
overall O |
|
structure B-evidence |
|
is O |
|
similar O |
|
to O |
|
that O |
|
of O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
, O |
|
although O |
|
there O |
|
are O |
|
also O |
|
substantial O |
|
conformational O |
|
differences O |
|
between O |
|
them O |
|
. O |
|
|
|
The O |
|
structure B-evidence |
|
reveals O |
|
a O |
|
surface B-site |
|
patch I-site |
|
that O |
|
is O |
|
conserved B-protein_state |
|
among O |
|
FPA B-protein |
|
homologs O |
|
. O |
|
|
|
Structure B-evidence |
|
of O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
|
|
The O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
of O |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
FPA B-protein |
|
has O |
|
been O |
|
determined O |
|
at O |
|
2 O |
|
. O |
|
7 O |
|
Å O |
|
resolution O |
|
using O |
|
the O |
|
selenomethionyl B-experimental_method |
|
single I-experimental_method |
|
- I-experimental_method |
|
wavelength I-experimental_method |
|
anomalous I-experimental_method |
|
dispersion I-experimental_method |
|
method I-experimental_method |
|
. O |
|
|
|
The O |
|
expression O |
|
construct O |
|
contained O |
|
residues O |
|
433 B-residue_range |
|
– I-residue_range |
|
565 I-residue_range |
|
of O |
|
FPA B-protein |
|
, O |
|
but O |
|
only O |
|
residues O |
|
439 B-residue_range |
|
– I-residue_range |
|
460 I-residue_range |
|
and O |
|
465 B-residue_range |
|
– I-residue_range |
|
565 I-residue_range |
|
are O |
|
ordered O |
|
in O |
|
the O |
|
crystal B-evidence |
|
. O |
|
|
|
The O |
|
atomic B-evidence |
|
model I-evidence |
|
has O |
|
good O |
|
agreement O |
|
with O |
|
the O |
|
X B-evidence |
|
- I-evidence |
|
ray I-evidence |
|
diffraction I-evidence |
|
data I-evidence |
|
and O |
|
the O |
|
expected O |
|
bond O |
|
lengths O |
|
, O |
|
bond O |
|
angles O |
|
and O |
|
other O |
|
geometric O |
|
parameters O |
|
( O |
|
Table O |
|
1 O |
|
). O |
|
|
|
All O |
|
the O |
|
residues O |
|
are O |
|
located O |
|
in O |
|
the O |
|
favored O |
|
regions O |
|
of O |
|
the O |
|
Ramachandran B-evidence |
|
plot I-evidence |
|
( O |
|
data O |
|
not O |
|
shown O |
|
). O |
|
|
|
The O |
|
structure B-evidence |
|
has O |
|
been O |
|
deposited O |
|
in O |
|
the O |
|
Protein O |
|
Data O |
|
Bank O |
|
, O |
|
with O |
|
accession O |
|
code O |
|
5KXF O |
|
. O |
|
|
|
Resolution O |
|
range O |
|
( O |
|
Å O |
|
) O |
|
1 O |
|
50 O |
|
– O |
|
2 O |
|
. O |
|
7 O |
|
( O |
|
2 O |
|
. O |
|
8 O |
|
– O |
|
2 O |
|
. O |
|
7 O |
|
) O |
|
Number O |
|
of O |
|
observations O |
|
78 O |
|
, O |
|
008 O |
|
Rmerge O |
|
(%) O |
|
10 O |
|
. O |
|
5 O |
|
( O |
|
45 O |
|
. O |
|
3 O |
|
) O |
|
I O |
|
/ O |
|
σI O |
|
24 O |
|
. O |
|
1 O |
|
( O |
|
6 O |
|
. O |
|
3 O |
|
) O |
|
Redundancy O |
|
Completeness O |
|
(%) O |
|
100 O |
|
( O |
|
100 O |
|
) O |
|
R B-evidence |
|
factor I-evidence |
|
(%) O |
|
19 O |
|
. O |
|
2 O |
|
( O |
|
25 O |
|
. O |
|
0 O |
|
) O |
|
Free B-evidence |
|
R I-evidence |
|
factor I-evidence |
|
(%) O |
|
25 O |
|
. O |
|
4 O |
|
( O |
|
35 O |
|
. O |
|
4 O |
|
) O |
|
Rms O |
|
deviation O |
|
in O |
|
bond O |
|
lengths O |
|
( O |
|
Å O |
|
) O |
|
0 O |
|
. O |
|
017 O |
|
Rms O |
|
deviation O |
|
in O |
|
bond O |
|
angles O |
|
(°) O |
|
1 O |
|
. O |
|
9 O |
|
|
|
The O |
|
crystal B-evidence |
|
structure I-evidence |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
contains O |
|
a O |
|
seven B-structure_element |
|
- I-structure_element |
|
stranded I-structure_element |
|
, I-structure_element |
|
mostly I-structure_element |
|
anti I-structure_element |
|
- I-structure_element |
|
parallel I-structure_element |
|
β I-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
( O |
|
β1 B-structure_element |
|
- I-structure_element |
|
β7 I-structure_element |
|
) O |
|
and O |
|
three O |
|
helices B-structure_element |
|
( O |
|
αA B-structure_element |
|
- I-structure_element |
|
αC I-structure_element |
|
) O |
|
( O |
|
Fig O |
|
2A O |
|
). O |
|
|
|
Only O |
|
two O |
|
of O |
|
the O |
|
neighboring O |
|
strands B-structure_element |
|
, O |
|
β1 B-structure_element |
|
and O |
|
β3 B-structure_element |
|
, O |
|
are O |
|
parallel O |
|
to O |
|
each O |
|
other O |
|
. O |
|
|
|
Helix B-structure_element |
|
αB B-structure_element |
|
covers O |
|
one O |
|
end O |
|
of O |
|
the O |
|
barrel B-structure_element |
|
, O |
|
while O |
|
helices B-structure_element |
|
αA B-structure_element |
|
and O |
|
αC B-structure_element |
|
are O |
|
located O |
|
next O |
|
to O |
|
each O |
|
other O |
|
at O |
|
one O |
|
side O |
|
of O |
|
the O |
|
barrel B-structure_element |
|
( O |
|
Fig O |
|
2B O |
|
). O |
|
|
|
The O |
|
other O |
|
end O |
|
of O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
is O |
|
covered O |
|
by O |
|
the O |
|
loop B-structure_element |
|
connecting O |
|
strands B-structure_element |
|
β2 B-structure_element |
|
and O |
|
β3 B-structure_element |
|
, O |
|
which O |
|
contains O |
|
the O |
|
disordered B-protein_state |
|
461 B-residue_range |
|
– I-residue_range |
|
464 I-residue_range |
|
segment O |
|
. O |
|
|
|
The O |
|
center O |
|
of O |
|
the O |
|
barrel B-structure_element |
|
is O |
|
filled O |
|
with O |
|
hydrophobic O |
|
side O |
|
chains O |
|
and O |
|
is O |
|
not O |
|
accessible O |
|
to O |
|
the O |
|
solvent O |
|
. O |
|
|
|
Crystal B-evidence |
|
structure I-evidence |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
of O |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
FPA B-protein |
|
. O |
|
|
|
Schematic O |
|
drawing O |
|
of O |
|
the O |
|
structure B-evidence |
|
of O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
, O |
|
colored O |
|
from O |
|
blue O |
|
at O |
|
the O |
|
N O |
|
terminus O |
|
to O |
|
red O |
|
at O |
|
the O |
|
C O |
|
terminus O |
|
. O |
|
|
|
The O |
|
view O |
|
is O |
|
from O |
|
the O |
|
side O |
|
of O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
. O |
|
|
|
The O |
|
disordered B-protein_state |
|
segment O |
|
( O |
|
residues O |
|
460 B-residue_range |
|
– I-residue_range |
|
465 I-residue_range |
|
) O |
|
is O |
|
indicated O |
|
with O |
|
the O |
|
dotted O |
|
line O |
|
. O |
|
|
|
Structure B-evidence |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
, O |
|
viewed O |
|
from O |
|
the O |
|
end O |
|
of O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
, O |
|
after O |
|
90 O |
|
° O |
|
rotation O |
|
around O |
|
the O |
|
horizontal O |
|
axis O |
|
from O |
|
panel O |
|
A O |
|
. O |
|
All O |
|
structure O |
|
figures O |
|
were O |
|
produced O |
|
with O |
|
PyMOL O |
|
( O |
|
www O |
|
. O |
|
pymol O |
|
. O |
|
org O |
|
). O |
|
|
|
Comparisons B-experimental_method |
|
to I-experimental_method |
|
structural I-experimental_method |
|
homologs I-experimental_method |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
|
|
Only O |
|
five O |
|
structural O |
|
homologs O |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
were O |
|
found O |
|
in O |
|
the O |
|
Protein O |
|
Data O |
|
Bank O |
|
with O |
|
the O |
|
DaliLite B-experimental_method |
|
server I-experimental_method |
|
, O |
|
suggesting O |
|
that O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
structure B-evidence |
|
is O |
|
relatively O |
|
unique O |
|
. O |
|
|
|
The O |
|
top O |
|
hit O |
|
is O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
of O |
|
human B-species |
|
SHARP B-protein |
|
( O |
|
Fig O |
|
3A O |
|
), O |
|
with O |
|
a O |
|
Z B-evidence |
|
score I-evidence |
|
of O |
|
12 O |
|
. O |
|
3 O |
|
. O |
|
|
|
The O |
|
other O |
|
four O |
|
structural O |
|
homologs O |
|
include O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
domain O |
|
of O |
|
the O |
|
proteins O |
|
Ku70 B-protein |
|
and O |
|
Ku80 B-protein |
|
( O |
|
Z B-evidence |
|
score I-evidence |
|
11 O |
|
. O |
|
4 O |
|
) O |
|
( O |
|
Fig O |
|
3B O |
|
), O |
|
a O |
|
domain O |
|
in O |
|
the O |
|
chromodomain B-protein_type |
|
protein I-protein_type |
|
Chp1 B-protein |
|
( O |
|
Z B-evidence |
|
score I-evidence |
|
10 O |
|
. O |
|
8 O |
|
) O |
|
( O |
|
Fig O |
|
3C O |
|
), O |
|
and O |
|
the O |
|
activator B-structure_element |
|
interacting I-structure_element |
|
domain I-structure_element |
|
( O |
|
ACID B-structure_element |
|
) O |
|
of O |
|
the O |
|
Med25 B-protein |
|
subunit O |
|
of O |
|
the O |
|
Mediator O |
|
complex O |
|
( O |
|
Z B-evidence |
|
score I-evidence |
|
8 O |
|
. O |
|
5 O |
|
) O |
|
( O |
|
Fig O |
|
3D O |
|
). O |
|
|
|
The O |
|
next O |
|
structural O |
|
homolog O |
|
has O |
|
a O |
|
Z B-evidence |
|
score I-evidence |
|
of O |
|
3 O |
|
. O |
|
0 O |
|
. O |
|
|
|
Structural O |
|
homologs O |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
. O |
|
|
|
Overlay B-experimental_method |
|
of O |
|
the O |
|
structures B-evidence |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
cyan O |
|
) O |
|
and O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
gray O |
|
). O |
|
|
|
The O |
|
bound O |
|
position O |
|
of O |
|
a O |
|
doubly B-protein_state |
|
- I-protein_state |
|
phosphorylated I-protein_state |
|
peptide B-chemical |
|
from O |
|
SMRT B-protein |
|
is O |
|
shown O |
|
in O |
|
magenta O |
|
. O |
|
|
|
Overlay B-experimental_method |
|
of O |
|
the O |
|
structures B-evidence |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
cyan O |
|
) O |
|
and O |
|
the O |
|
Ku70 B-protein |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
domain O |
|
( O |
|
gray O |
|
). O |
|
|
|
Ku80 B-protein |
|
contains O |
|
a O |
|
homologous O |
|
domain O |
|
( O |
|
green O |
|
), O |
|
which O |
|
forms O |
|
a O |
|
hetero B-oligomeric_state |
|
- I-oligomeric_state |
|
dimer I-oligomeric_state |
|
with O |
|
that O |
|
in O |
|
Ku70 B-protein |
|
. O |
|
|
|
The O |
|
two O |
|
domains O |
|
, O |
|
and O |
|
inserted O |
|
segments O |
|
on O |
|
them O |
|
, O |
|
mediate O |
|
the O |
|
binding O |
|
of O |
|
dsDNA B-chemical |
|
( O |
|
orange O |
|
). O |
|
|
|
The O |
|
red O |
|
rectangle O |
|
highlights O |
|
the O |
|
region O |
|
of O |
|
contact O |
|
between O |
|
the O |
|
two O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
domains O |
|
. O |
|
|
|
Overlay B-experimental_method |
|
of O |
|
the O |
|
structures B-evidence |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
cyan O |
|
) O |
|
and O |
|
the O |
|
homologous O |
|
domain O |
|
in O |
|
Chp1 B-protein |
|
( O |
|
gray O |
|
). O |
|
|
|
The O |
|
binding O |
|
partner O |
|
of O |
|
Chp1 B-protein |
|
, O |
|
Tas3 B-protein |
|
, O |
|
is O |
|
shown O |
|
in O |
|
green O |
|
. O |
|
|
|
The O |
|
red O |
|
rectangle O |
|
indicates O |
|
the O |
|
region O |
|
equivalent O |
|
to O |
|
the O |
|
binding B-site |
|
site I-site |
|
of O |
|
the O |
|
SMART B-protein |
|
phosphopeptide B-ptm |
|
in O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
, O |
|
where O |
|
a O |
|
loop B-structure_element |
|
of O |
|
Tas3 B-protein |
|
is O |
|
also O |
|
located O |
|
. O |
|
( O |
|
D O |
|
). O |
|
|
|
Overlay B-experimental_method |
|
of O |
|
the O |
|
structures B-evidence |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
cyan O |
|
) O |
|
and O |
|
the O |
|
Med25 B-protein |
|
ACID B-structure_element |
|
( O |
|
gray O |
|
). O |
|
|
|
SHARP B-protein |
|
is O |
|
a O |
|
transcriptional B-protein_type |
|
co I-protein_type |
|
- I-protein_type |
|
repressor I-protein_type |
|
in O |
|
the O |
|
nuclear B-protein_type |
|
receptor I-protein_type |
|
and O |
|
Notch B-protein |
|
/ O |
|
RBP B-protein |
|
- I-protein |
|
Jκ I-protein |
|
signaling O |
|
pathways O |
|
. O |
|
|
|
The O |
|
SPOC B-structure_element |
|
domain O |
|
of O |
|
SHARP B-protein |
|
interacts O |
|
directly O |
|
with O |
|
silencing B-protein |
|
mediator I-protein |
|
for I-protein |
|
retinoid I-protein |
|
and I-protein |
|
thyroid I-protein |
|
receptor I-protein |
|
( O |
|
SMRT B-protein |
|
), O |
|
nuclear B-protein_type |
|
receptor I-protein_type |
|
co I-protein_type |
|
- I-protein_type |
|
repressor I-protein_type |
|
( O |
|
N B-protein_type |
|
- I-protein_type |
|
CoR I-protein_type |
|
), O |
|
HDAC B-protein |
|
, O |
|
and O |
|
other O |
|
components O |
|
to O |
|
represses O |
|
transcription O |
|
. O |
|
|
|
While O |
|
the O |
|
overall O |
|
structure B-evidence |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
is O |
|
similar O |
|
to O |
|
that O |
|
of O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
, O |
|
there O |
|
are O |
|
noticeable O |
|
differences O |
|
in O |
|
the O |
|
positioning O |
|
of O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
strands I-structure_element |
|
and O |
|
the O |
|
helices B-structure_element |
|
, O |
|
and O |
|
most O |
|
of O |
|
the O |
|
loops B-structure_element |
|
have O |
|
substantially O |
|
different O |
|
conformations O |
|
as O |
|
well O |
|
( O |
|
Fig O |
|
3A O |
|
). O |
|
|
|
In O |
|
addition O |
|
, O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
has O |
|
three O |
|
extra O |
|
helices B-structure_element |
|
. O |
|
|
|
One O |
|
of O |
|
them O |
|
covers O |
|
the O |
|
other O |
|
end O |
|
of O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
, O |
|
and O |
|
the O |
|
other O |
|
two O |
|
shield O |
|
an O |
|
additional O |
|
surface O |
|
of O |
|
the O |
|
side O |
|
of O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
from O |
|
solvent O |
|
. O |
|
|
|
A O |
|
doubly B-protein_state |
|
- I-protein_state |
|
phosphorylated I-protein_state |
|
peptide B-chemical |
|
from O |
|
SMRT B-protein |
|
is O |
|
bound B-protein_state |
|
to I-protein_state |
|
the O |
|
side O |
|
of O |
|
the O |
|
barrel B-structure_element |
|
, O |
|
near O |
|
strands B-structure_element |
|
β1 B-structure_element |
|
and O |
|
β3 B-structure_element |
|
( O |
|
Fig O |
|
3A O |
|
). O |
|
|
|
Such O |
|
a O |
|
binding O |
|
mode O |
|
probably O |
|
would O |
|
not O |
|
be O |
|
possible O |
|
in O |
|
FPA B-protein |
|
, O |
|
as O |
|
the O |
|
peptide B-chemical |
|
would O |
|
clash O |
|
with O |
|
the O |
|
β1 B-structure_element |
|
- I-structure_element |
|
β2 I-structure_element |
|
loop I-structure_element |
|
. O |
|
|
|
The O |
|
Ku70 B-complex_assembly |
|
- I-complex_assembly |
|
Ku80 I-complex_assembly |
|
hetero B-oligomeric_state |
|
- I-oligomeric_state |
|
dimer I-oligomeric_state |
|
is O |
|
involved O |
|
in O |
|
DNA O |
|
double O |
|
- O |
|
strand O |
|
break O |
|
repair O |
|
and O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
domain O |
|
contributes O |
|
to O |
|
DNA B-chemical |
|
binding O |
|
. O |
|
|
|
In O |
|
fact O |
|
, O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
domains O |
|
of O |
|
Ku70 B-protein |
|
and O |
|
Ku80 B-protein |
|
form O |
|
a O |
|
hetero B-oligomeric_state |
|
- I-oligomeric_state |
|
dimer I-oligomeric_state |
|
, O |
|
primarily O |
|
through O |
|
interactions O |
|
between O |
|
the O |
|
loops B-structure_element |
|
connecting O |
|
the O |
|
third B-structure_element |
|
and I-structure_element |
|
fourth I-structure_element |
|
strands I-structure_element |
|
of O |
|
the O |
|
barrel B-structure_element |
|
( O |
|
Fig O |
|
3B O |
|
). O |
|
|
|
The O |
|
open O |
|
ends O |
|
of O |
|
the O |
|
two O |
|
β B-structure_element |
|
- I-structure_element |
|
barrels I-structure_element |
|
face O |
|
the O |
|
DNA B-site |
|
binding I-site |
|
sites I-site |
|
, O |
|
and O |
|
contact O |
|
the O |
|
phosphodiester O |
|
backbone O |
|
of O |
|
the O |
|
dsDNA B-chemical |
|
. O |
|
|
|
In O |
|
addition O |
|
, O |
|
a O |
|
long B-structure_element |
|
insert I-structure_element |
|
connecting O |
|
strands B-structure_element |
|
β2 B-structure_element |
|
and O |
|
β3 B-structure_element |
|
in O |
|
the O |
|
two O |
|
domains O |
|
form O |
|
an O |
|
arch B-structure_element |
|
- I-structure_element |
|
like I-structure_element |
|
structure I-structure_element |
|
, O |
|
encircling O |
|
the O |
|
dsDNA B-chemical |
|
. O |
|
|
|
Chp1 B-protein |
|
is O |
|
a O |
|
subunit O |
|
of O |
|
the O |
|
RNA B-complex_assembly |
|
- I-complex_assembly |
|
induced I-complex_assembly |
|
initiation I-complex_assembly |
|
of I-complex_assembly |
|
transcriptional I-complex_assembly |
|
gene I-complex_assembly |
|
silencing I-complex_assembly |
|
( O |
|
RITS B-complex_assembly |
|
) O |
|
complex O |
|
. O |
|
|
|
The O |
|
partner O |
|
of O |
|
Chp1 B-protein |
|
, O |
|
Tas3 B-protein |
|
, O |
|
is O |
|
bound O |
|
between O |
|
the O |
|
barrel B-structure_element |
|
domain I-structure_element |
|
and O |
|
the O |
|
second B-structure_element |
|
domain I-structure_element |
|
of O |
|
Chp1 B-protein |
|
, O |
|
and O |
|
the O |
|
linker B-structure_element |
|
between O |
|
the O |
|
two O |
|
domains O |
|
is O |
|
also O |
|
crucial O |
|
for O |
|
this O |
|
interaction O |
|
( O |
|
Fig O |
|
3C O |
|
). O |
|
|
|
It O |
|
is O |
|
probably O |
|
unlikely O |
|
that O |
|
the O |
|
β B-structure_element |
|
- I-structure_element |
|
barrel I-structure_element |
|
itself O |
|
is O |
|
sufficient O |
|
to O |
|
bind O |
|
Tas3 B-protein |
|
. O |
|
|
|
Interestingly O |
|
, O |
|
a O |
|
loop B-structure_element |
|
in O |
|
Tas3 B-protein |
|
contacts O |
|
strand B-structure_element |
|
β3 B-structure_element |
|
of O |
|
the O |
|
barrel B-structure_element |
|
domain I-structure_element |
|
, O |
|
at O |
|
a O |
|
location O |
|
somewhat O |
|
similar O |
|
to O |
|
that O |
|
of O |
|
the O |
|
N O |
|
- O |
|
terminal O |
|
segment O |
|
of O |
|
the O |
|
SMRT B-protein |
|
peptide B-chemical |
|
in B-protein_state |
|
complex I-protein_state |
|
with I-protein_state |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
Fig O |
|
3A O |
|
). O |
|
|
|
Mediator B-protein_type |
|
is O |
|
a O |
|
coactivator O |
|
complex O |
|
that O |
|
promotes O |
|
transcription O |
|
by O |
|
Pol B-complex_assembly |
|
II I-complex_assembly |
|
. O |
|
|
|
The O |
|
Med25 B-protein |
|
subunit O |
|
ACID B-structure_element |
|
is O |
|
the O |
|
target O |
|
of O |
|
the O |
|
potent O |
|
activator O |
|
VP16 B-protein |
|
of O |
|
the O |
|
herpes B-species |
|
simplex I-species |
|
virus I-species |
|
. O |
|
|
|
The O |
|
structure B-evidence |
|
of O |
|
ACID B-structure_element |
|
contains O |
|
a O |
|
helix B-structure_element |
|
at O |
|
the O |
|
C O |
|
- O |
|
terminus O |
|
as O |
|
well O |
|
as O |
|
an O |
|
extended O |
|
β1 B-structure_element |
|
- I-structure_element |
|
β2 I-structure_element |
|
loop I-structure_element |
|
. O |
|
|
|
Nonetheless O |
|
, O |
|
the O |
|
binding B-site |
|
site I-site |
|
for O |
|
VP16 B-protein |
|
has O |
|
been O |
|
mapped O |
|
to O |
|
roughly O |
|
the O |
|
same O |
|
surface B-site |
|
patch I-site |
|
, O |
|
near O |
|
strands B-structure_element |
|
β1 B-structure_element |
|
and O |
|
β3 B-structure_element |
|
, O |
|
that O |
|
is O |
|
used O |
|
by O |
|
the O |
|
SHARP B-protein |
|
and O |
|
Tas3 B-protein |
|
SPOC B-structure_element |
|
domains O |
|
for O |
|
binding O |
|
their O |
|
partners O |
|
. O |
|
|
|
A O |
|
conserved B-protein_state |
|
surface B-site |
|
patch I-site |
|
in O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
|
|
An O |
|
analysis O |
|
of O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
indicates O |
|
a O |
|
large O |
|
surface B-site |
|
patch I-site |
|
near O |
|
strands B-structure_element |
|
β1 B-structure_element |
|
, O |
|
β3 B-structure_element |
|
, O |
|
β5 B-structure_element |
|
and O |
|
β6 B-structure_element |
|
that O |
|
is O |
|
conserved B-protein_state |
|
among O |
|
plant B-taxonomy_domain |
|
FPA B-protein |
|
homologs O |
|
( O |
|
Fig O |
|
4A O |
|
). O |
|
|
|
This O |
|
surface B-site |
|
patch I-site |
|
can O |
|
be O |
|
broken O |
|
into O |
|
two O |
|
sub B-site |
|
- I-site |
|
patches I-site |
|
, O |
|
with O |
|
residues O |
|
Lys447 B-residue_name_number |
|
( O |
|
in O |
|
strand B-structure_element |
|
β1 B-structure_element |
|
), O |
|
Arg477 B-residue_name_number |
|
( O |
|
β3 B-structure_element |
|
), O |
|
Tyr515 B-residue_name_number |
|
( O |
|
αB B-structure_element |
|
) O |
|
and O |
|
Arg521 B-residue_name_number |
|
( O |
|
β5 B-structure_element |
|
) O |
|
in O |
|
one O |
|
sub B-site |
|
- I-site |
|
patch I-site |
|
, O |
|
and O |
|
residues O |
|
His486 B-residue_name_number |
|
( O |
|
αA B-structure_element |
|
), O |
|
Thr478 B-residue_name_number |
|
( O |
|
β3 B-structure_element |
|
), O |
|
Val524 B-residue_name_number |
|
( O |
|
β5 B-structure_element |
|
) O |
|
and O |
|
Phe534 B-residue_name_number |
|
( O |
|
β6 B-structure_element |
|
) O |
|
in O |
|
the O |
|
other O |
|
sub B-site |
|
- I-site |
|
patch I-site |
|
( O |
|
Fig O |
|
4B O |
|
). O |
|
|
|
The O |
|
first B-site |
|
surface I-site |
|
patch I-site |
|
is O |
|
electropositive B-protein_state |
|
in O |
|
nature O |
|
( O |
|
Fig O |
|
4C O |
|
), O |
|
and O |
|
residues O |
|
Arg477 B-residue_name_number |
|
and O |
|
Tyr515 B-residue_name_number |
|
are O |
|
also O |
|
conserved B-protein_state |
|
in O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
Fig O |
|
1B O |
|
). O |
|
|
|
In O |
|
fact O |
|
, O |
|
one O |
|
of O |
|
the O |
|
phosphorylated B-protein_state |
|
residues O |
|
of O |
|
the O |
|
SMRT B-protein |
|
peptide B-chemical |
|
interacts O |
|
with O |
|
this O |
|
surface B-site |
|
patch I-site |
|
( O |
|
Fig O |
|
3A O |
|
), O |
|
suggesting O |
|
that O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
might O |
|
also O |
|
interact O |
|
with O |
|
a O |
|
phosphorylated B-protein_state |
|
segment O |
|
here O |
|
. O |
|
|
|
In O |
|
comparison O |
|
, O |
|
the O |
|
second B-site |
|
surface I-site |
|
patch I-site |
|
is O |
|
more O |
|
hydrophobic B-protein_state |
|
in O |
|
nature O |
|
( O |
|
Fig O |
|
4C O |
|
). O |
|
|
|
A O |
|
conserved B-protein_state |
|
surface B-site |
|
patch I-site |
|
of O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
. O |
|
|
|
Two O |
|
views O |
|
of O |
|
the O |
|
molecular O |
|
surface O |
|
of O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
colored O |
|
based O |
|
on O |
|
sequence O |
|
conservation O |
|
among O |
|
plant B-taxonomy_domain |
|
FPA B-protein |
|
homologs O |
|
. O |
|
|
|
Residues O |
|
in O |
|
the O |
|
conserved B-protein_state |
|
surface B-site |
|
patch I-site |
|
of O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
. O |
|
|
|
The O |
|
side O |
|
chains O |
|
of O |
|
the O |
|
residues O |
|
are O |
|
shown O |
|
in O |
|
stick O |
|
models O |
|
, O |
|
colored O |
|
orange O |
|
in O |
|
the O |
|
first B-site |
|
sub I-site |
|
- I-site |
|
patch I-site |
|
and O |
|
green O |
|
in O |
|
the O |
|
second O |
|
. O |
|
( O |
|
C O |
|
). O |
|
|
|
Molecular O |
|
surface O |
|
of O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
colored O |
|
based O |
|
on O |
|
electrostatic O |
|
potential O |
|
. O |
|
|
|
Testing O |
|
the O |
|
requirement O |
|
of O |
|
specific O |
|
conserved O |
|
amino O |
|
acids O |
|
for O |
|
FPA B-protein |
|
functions O |
|
|
|
We O |
|
next O |
|
examined O |
|
the O |
|
potential O |
|
impact O |
|
of O |
|
the O |
|
conserved B-protein_state |
|
surface B-site |
|
patch I-site |
|
on O |
|
FPA B-protein |
|
function O |
|
in O |
|
vivo O |
|
. O |
|
|
|
We O |
|
mutated B-experimental_method |
|
two O |
|
residues O |
|
, O |
|
Arg477 B-residue_name_number |
|
and O |
|
Tyr515 B-residue_name_number |
|
, O |
|
of O |
|
the O |
|
surface B-site |
|
patch I-site |
|
, O |
|
which O |
|
are O |
|
also O |
|
conserved B-protein_state |
|
in O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
( O |
|
Fig O |
|
1B O |
|
) O |
|
and O |
|
were O |
|
found O |
|
to O |
|
be O |
|
functionally O |
|
important O |
|
. O |
|
|
|
The O |
|
mutations B-experimental_method |
|
were O |
|
introduced B-experimental_method |
|
into O |
|
a O |
|
transgene O |
|
designed O |
|
to O |
|
express O |
|
FPA B-protein |
|
from O |
|
its O |
|
native O |
|
control O |
|
elements O |
|
( O |
|
promoter O |
|
, O |
|
introns O |
|
and O |
|
3 O |
|
′ O |
|
UTR O |
|
). O |
|
|
|
The O |
|
resulting O |
|
transgenes O |
|
were O |
|
then O |
|
stably B-experimental_method |
|
transformed I-experimental_method |
|
into O |
|
an O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
mutant B-protein_state |
|
background O |
|
so O |
|
that O |
|
the O |
|
impact O |
|
of O |
|
the O |
|
mutations B-experimental_method |
|
on O |
|
FPA B-protein |
|
function O |
|
could O |
|
be O |
|
assessed O |
|
. O |
|
|
|
Control O |
|
transformation O |
|
of O |
|
the O |
|
same O |
|
expression B-experimental_method |
|
constructs I-experimental_method |
|
into O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
designed O |
|
to O |
|
express O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
FPA B-protein |
|
protein O |
|
restored O |
|
FPA B-protein |
|
protein O |
|
expression B-evidence |
|
levels I-evidence |
|
to O |
|
near O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
levels O |
|
( O |
|
panel O |
|
A O |
|
in O |
|
S1 O |
|
Fig O |
|
) O |
|
and O |
|
rescued O |
|
the O |
|
function O |
|
of O |
|
FPA B-protein |
|
in O |
|
controlling O |
|
RNA B-chemical |
|
3 O |
|
′- O |
|
end O |
|
formation O |
|
, O |
|
for O |
|
example O |
|
in O |
|
FPA B-protein |
|
pre B-chemical |
|
- I-chemical |
|
mRNA I-chemical |
|
( O |
|
panel O |
|
B O |
|
in O |
|
S1 O |
|
Fig O |
|
). O |
|
|
|
We O |
|
examined O |
|
independent O |
|
transgenic O |
|
lines O |
|
expressing O |
|
each O |
|
R477A B-mutant |
|
and O |
|
Y515A B-mutant |
|
mutation B-experimental_method |
|
. O |
|
|
|
In O |
|
each O |
|
case O |
|
, O |
|
we O |
|
confirmed O |
|
that O |
|
detectable O |
|
levels O |
|
of O |
|
FPA B-protein |
|
protein O |
|
expression O |
|
were O |
|
restored O |
|
close O |
|
to O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
levels O |
|
in O |
|
protein B-experimental_method |
|
blot I-experimental_method |
|
analyses O |
|
using O |
|
antibodies O |
|
that O |
|
specifically O |
|
recognize O |
|
FPA B-protein |
|
( O |
|
S2 O |
|
Fig O |
|
). O |
|
|
|
We O |
|
then O |
|
examined O |
|
the O |
|
impact O |
|
of O |
|
the O |
|
surface B-site |
|
patch I-site |
|
mutations B-experimental_method |
|
on O |
|
FPA B-protein |
|
’ O |
|
s O |
|
function O |
|
in O |
|
controlling O |
|
RNA O |
|
3 O |
|
′- O |
|
end O |
|
formation O |
|
by O |
|
determining O |
|
whether O |
|
the O |
|
mutant B-protein_state |
|
proteins O |
|
functioned O |
|
in O |
|
FPA B-protein |
|
autoregulation O |
|
and O |
|
the O |
|
repression O |
|
of O |
|
FLC B-gene |
|
expression O |
|
. O |
|
|
|
FPA B-protein |
|
autoregulates O |
|
its O |
|
expression O |
|
by O |
|
promoting O |
|
cleavage O |
|
and O |
|
polyadenylation O |
|
within O |
|
intron O |
|
1 O |
|
of O |
|
its O |
|
own O |
|
pre B-chemical |
|
- I-chemical |
|
mRNA I-chemical |
|
, O |
|
resulting O |
|
in O |
|
a O |
|
truncated O |
|
transcript O |
|
that O |
|
does O |
|
not O |
|
encode O |
|
functional O |
|
protein O |
|
. O |
|
|
|
We O |
|
used O |
|
RNA B-experimental_method |
|
gel I-experimental_method |
|
blot I-experimental_method |
|
analyses I-experimental_method |
|
to O |
|
reveal O |
|
that O |
|
in O |
|
each O |
|
of O |
|
three O |
|
independent O |
|
transgenic O |
|
lines O |
|
for O |
|
each O |
|
single O |
|
mutant B-protein_state |
|
, O |
|
rescue O |
|
of O |
|
proximally O |
|
polyadenylated O |
|
FPA B-protein |
|
pre B-chemical |
|
- I-chemical |
|
mRNA I-chemical |
|
can O |
|
be O |
|
detected O |
|
( O |
|
Fig O |
|
5A O |
|
and O |
|
5B O |
|
). O |
|
|
|
We O |
|
therefore O |
|
conclude O |
|
that O |
|
neither O |
|
of O |
|
these O |
|
mutations O |
|
disrupted O |
|
the O |
|
ability O |
|
of O |
|
FPA B-protein |
|
to O |
|
promote O |
|
RNA O |
|
3 O |
|
′- O |
|
end O |
|
formation O |
|
in O |
|
its O |
|
own O |
|
transcript O |
|
. O |
|
|
|
Impact O |
|
of O |
|
individual O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
mutations B-experimental_method |
|
on O |
|
alternative O |
|
polyadenylation O |
|
of O |
|
FPA B-protein |
|
pre B-chemical |
|
- I-chemical |
|
mRNA I-chemical |
|
. O |
|
|
|
RNA B-experimental_method |
|
gel I-experimental_method |
|
blot I-experimental_method |
|
analysis O |
|
of O |
|
WT B-protein_state |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
accession O |
|
Columbia O |
|
( O |
|
Col O |
|
- O |
|
0 O |
|
) O |
|
plants B-taxonomy_domain |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
and O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
mutants B-protein_state |
|
expressing O |
|
either O |
|
FPA B-protein |
|
:: O |
|
FPA B-mutant |
|
R477A I-mutant |
|
( O |
|
A O |
|
), O |
|
or O |
|
FPA B-protein |
|
:: O |
|
FPA B-mutant |
|
Y515A I-mutant |
|
( O |
|
B O |
|
) O |
|
using O |
|
poly O |
|
( O |
|
A O |
|
)+ O |
|
purified O |
|
mRNAs B-chemical |
|
. O |
|
|
|
A O |
|
probe O |
|
corresponding O |
|
to O |
|
the O |
|
5 O |
|
’ O |
|
UTR O |
|
region O |
|
of O |
|
FPA B-protein |
|
mRNA B-chemical |
|
was O |
|
used O |
|
to O |
|
detect O |
|
FPA B-protein |
|
specific O |
|
mRNAs B-chemical |
|
. O |
|
|
|
Proximally O |
|
and O |
|
distally O |
|
polyadenylated O |
|
FPA B-protein |
|
transcripts O |
|
are O |
|
marked O |
|
with O |
|
arrows O |
|
. O |
|
|
|
The O |
|
ratio O |
|
of O |
|
distal O |
|
: O |
|
proximal O |
|
polyadenylated O |
|
forms O |
|
is O |
|
given O |
|
under O |
|
each O |
|
lane O |
|
. O |
|
( O |
|
C O |
|
, O |
|
D O |
|
) O |
|
Impact O |
|
of O |
|
individual O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
mutations B-experimental_method |
|
on O |
|
FLC B-gene |
|
transcript O |
|
levels O |
|
. O |
|
|
|
qRT B-experimental_method |
|
- I-experimental_method |
|
PCR I-experimental_method |
|
analysis O |
|
was O |
|
performed O |
|
with O |
|
total O |
|
RNA B-chemical |
|
purified O |
|
from O |
|
Col O |
|
- O |
|
0 O |
|
, O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
, O |
|
35S O |
|
:: O |
|
FPA B-protein |
|
: O |
|
YFP B-experimental_method |
|
and O |
|
FPA B-protein |
|
:: O |
|
FPA B-mutant |
|
R477A I-mutant |
|
( O |
|
C O |
|
), O |
|
FPA B-protein |
|
:: O |
|
FPA B-mutant |
|
Y515A I-mutant |
|
( O |
|
D O |
|
) O |
|
plants B-taxonomy_domain |
|
. O |
|
|
|
Histograms B-evidence |
|
show O |
|
mean O |
|
values O |
|
± O |
|
SE O |
|
for O |
|
three O |
|
independent O |
|
PCR B-experimental_method |
|
amplifications O |
|
of O |
|
three O |
|
biological O |
|
replicates O |
|
. O |
|
|
|
We O |
|
next O |
|
examined O |
|
whether O |
|
the O |
|
corresponding O |
|
mutations O |
|
disrupted O |
|
the O |
|
ability O |
|
of O |
|
FPA B-protein |
|
to O |
|
control O |
|
FLC B-gene |
|
expression O |
|
. O |
|
|
|
We O |
|
used O |
|
RT B-experimental_method |
|
- I-experimental_method |
|
qPCR I-experimental_method |
|
to O |
|
measure O |
|
the O |
|
expression O |
|
of O |
|
FLC B-gene |
|
mRNA B-chemical |
|
and O |
|
found O |
|
that O |
|
in O |
|
each O |
|
independent O |
|
transgenic O |
|
line O |
|
encoding O |
|
each O |
|
mutated B-protein_state |
|
FPA B-protein |
|
protein O |
|
, O |
|
the O |
|
elevated O |
|
levels O |
|
of O |
|
FLC B-gene |
|
detected O |
|
in O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
mutants B-protein_state |
|
were O |
|
restored O |
|
to O |
|
near O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
levels O |
|
by O |
|
expression O |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
conserved B-protein_state |
|
patch B-site |
|
mutant B-protein_state |
|
proteins O |
|
( O |
|
Fig O |
|
5C O |
|
and O |
|
5D O |
|
). O |
|
|
|
Since O |
|
each O |
|
surface B-site |
|
patch I-site |
|
mutation B-experimental_method |
|
appeared O |
|
to O |
|
be O |
|
insufficient O |
|
to O |
|
disrupt O |
|
FPA B-protein |
|
functions O |
|
on O |
|
its O |
|
own O |
|
, O |
|
we O |
|
combined O |
|
both O |
|
mutations O |
|
into O |
|
the O |
|
same O |
|
transgene O |
|
. O |
|
|
|
We O |
|
could O |
|
again O |
|
confirm O |
|
that O |
|
near O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
levels O |
|
of O |
|
FPA B-protein |
|
protein O |
|
were O |
|
expressed O |
|
from O |
|
three O |
|
independent O |
|
transgenic O |
|
lines O |
|
expressing O |
|
the O |
|
FPA B-mutant |
|
R477A I-mutant |
|
; I-mutant |
|
Y515A I-mutant |
|
doubly B-protein_state |
|
mutated I-protein_state |
|
protein O |
|
in O |
|
an O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
mutant B-protein_state |
|
background O |
|
( O |
|
S3 O |
|
Fig O |
|
). O |
|
|
|
We O |
|
found O |
|
that O |
|
FPA B-mutant |
|
R477A I-mutant |
|
; I-mutant |
|
Y515A I-mutant |
|
protein O |
|
functioned O |
|
like O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
FPA B-protein |
|
to O |
|
restore O |
|
FPA B-protein |
|
pre B-chemical |
|
- I-chemical |
|
mRNA I-chemical |
|
proximal O |
|
polyadenylation O |
|
( O |
|
Fig O |
|
6A O |
|
) O |
|
and O |
|
FLC B-gene |
|
expression O |
|
to O |
|
wild B-protein_state |
|
- I-protein_state |
|
type I-protein_state |
|
levels O |
|
( O |
|
Fig O |
|
6B O |
|
). O |
|
|
|
Impact O |
|
of O |
|
double O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
mutations B-experimental_method |
|
on O |
|
alternative O |
|
polyadenylation O |
|
of O |
|
FPA B-protein |
|
pre B-chemical |
|
- I-chemical |
|
mRNA I-chemical |
|
and O |
|
FLC B-gene |
|
expression O |
|
. O |
|
|
|
( O |
|
A O |
|
) O |
|
RNA B-experimental_method |
|
gel I-experimental_method |
|
blot I-experimental_method |
|
analysis O |
|
of O |
|
WT B-protein_state |
|
A B-species |
|
. I-species |
|
thaliana I-species |
|
accession O |
|
Columbia O |
|
( O |
|
Col O |
|
- O |
|
0 O |
|
) O |
|
plants B-taxonomy_domain |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
and O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
mutants B-protein_state |
|
expressing O |
|
FPA B-protein |
|
:: O |
|
FPA B-mutant |
|
R477A I-mutant |
|
; I-mutant |
|
Y515A I-mutant |
|
using O |
|
poly O |
|
( O |
|
A O |
|
)+ O |
|
purified O |
|
mRNAs B-chemical |
|
. O |
|
|
|
Black O |
|
arrows O |
|
indicate O |
|
the O |
|
proximally O |
|
and O |
|
distally O |
|
polyadenylated O |
|
FPA B-protein |
|
mRNAs B-chemical |
|
. O |
|
|
|
qRT B-experimental_method |
|
- I-experimental_method |
|
PCR I-experimental_method |
|
analysis O |
|
was O |
|
performed O |
|
with O |
|
total O |
|
RNA B-chemical |
|
purified O |
|
from O |
|
Col O |
|
- O |
|
0 O |
|
, O |
|
fpa B-gene |
|
- I-gene |
|
8 I-gene |
|
, O |
|
and O |
|
FPA B-protein |
|
:: O |
|
FPA B-mutant |
|
R477A I-mutant |
|
; I-mutant |
|
Y515A I-mutant |
|
plants B-taxonomy_domain |
|
. O |
|
|
|
Together O |
|
our O |
|
findings O |
|
suggest O |
|
that O |
|
either O |
|
the O |
|
SPOC B-structure_element |
|
domain O |
|
is O |
|
not O |
|
required O |
|
for O |
|
the O |
|
role O |
|
of O |
|
FPA B-protein |
|
in O |
|
regulating O |
|
RNA B-chemical |
|
3 O |
|
′- O |
|
end O |
|
formation O |
|
, O |
|
or O |
|
that O |
|
this O |
|
combination O |
|
of O |
|
mutations B-experimental_method |
|
is O |
|
not O |
|
sufficient O |
|
to O |
|
critically O |
|
disrupt O |
|
the O |
|
function O |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
. O |
|
|
|
Since O |
|
the O |
|
corresponding O |
|
mutations B-experimental_method |
|
in O |
|
the O |
|
SHARP B-protein |
|
SPOC B-structure_element |
|
domain O |
|
do O |
|
disrupt O |
|
its O |
|
recognition O |
|
of O |
|
unphosphorylated B-protein_state |
|
SMRT B-protein |
|
peptides B-chemical |
|
, O |
|
these O |
|
observations O |
|
may O |
|
reinforce O |
|
the O |
|
idea O |
|
that O |
|
the O |
|
features O |
|
and O |
|
functions O |
|
of O |
|
the O |
|
FPA B-protein |
|
SPOC B-structure_element |
|
domain O |
|
differ O |
|
from O |
|
those O |
|
of O |
|
the O |
|
only O |
|
other O |
|
well O |
|
- O |
|
characterized O |
|
SPOC B-structure_element |
|
domain O |
|
. O |
|
|
|
|