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