Roquin	B-protein
recognizes	O
a	O
non	O
-	O
canonical	O
hexaloop	B-structure_element
structure	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Ox40	B-protein

The	O
RNA	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
Roquin	B-protein
is	O
required	O
to	O
prevent	O
autoimmunity	O
.	O

Roquin	B-protein
controls	O
T	O
-	O
helper	O
cell	O
activation	O
and	O
differentiation	O
by	O
limiting	O
the	O
induced	O
expression	O
of	O
costimulatory	B-protein_type
receptors	I-protein_type
such	O
as	O
tumor	B-protein
necrosis	I-protein
factor	I-protein
receptor	I-protein
superfamily	I-protein
4	I-protein
(	O
Tnfrs4	B-protein
or	O
Ox40	B-protein
).	O

A	O
constitutive	B-structure_element
decay	I-structure_element
element	I-structure_element
(	O
CDE	B-structure_element
)	O
with	O
a	O
characteristic	O
triloop	B-structure_element
hairpin	I-structure_element
was	O
previously	O
shown	O
to	O
be	O
recognized	O
by	O
Roquin	B-protein
.	O

Here	O
we	O
use	O
SELEX	B-experimental_method
assays	I-experimental_method
to	O
identify	O
a	O
novel	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
hexaloop	I-structure_element
motif	I-structure_element
,	O
representing	O
an	O
alternative	B-structure_element
decay	I-structure_element
element	I-structure_element
(	O
ADE	B-structure_element
).	O

Crystal	B-evidence
structures	I-evidence
and	O
NMR	B-experimental_method
data	O
show	O
that	O
the	O
Roquin	B-protein
-	I-protein
1	I-protein
ROQ	B-structure_element
domain	O
recognizes	O
hexaloops	B-structure_element
in	O
the	O
SELEX	B-experimental_method
-	O
derived	O
ADE	B-structure_element
and	O
in	O
an	O
ADE	B-structure_element
-	O
like	O
variant	O
present	O
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
with	O
identical	O
binding	O
modes	O
.	O

In	O
cells	O
,	O
ADE	B-structure_element
-	O
like	O
and	O
CDE	B-structure_element
-	O
like	O
motifs	O
cooperate	O
in	O
the	O
repression	O
of	O
Ox40	B-protein
by	O
Roquin	B-protein
.	O

Our	O
data	O
reveal	O
an	O
unexpected	O
recognition	O
of	O
hexaloop	B-structure_element
cis	I-structure_element
elements	I-structure_element
for	O
the	O
posttranscriptional	O
regulation	O
of	O
target	O
messenger	B-chemical
RNAs	I-chemical
by	O
Roquin	B-protein
.	O

Roquin	B-protein
is	O
an	O
RNA	B-protein_type
-	I-protein_type
binding	I-protein_type
protein	I-protein_type
that	O
prevents	O
autoimmunity	O
by	O
limiting	O
expression	O
of	O
receptors	O
such	O
as	O
Ox40	B-protein
.	O

Here	O
,	O
the	O
authors	O
identify	O
an	O
RNA	B-chemical
structure	B-evidence
that	O
they	O
describe	O
as	O
an	O
alternative	B-structure_element
decay	I-structure_element
element	I-structure_element
,	O
and	O
they	O
characterise	O
its	O
interaction	O
with	O
Roquin	B-protein
using	O
structural	B-experimental_method
and	I-experimental_method
biochemical	I-experimental_method
techniques	I-experimental_method
.	O

The	O
Roquin	B-protein
protein	O
is	O
essential	O
in	O
T	O
cells	O
for	O
the	O
prevention	O
of	O
autoimmune	O
disease	O
.	O

This	O
is	O
evident	O
from	O
the	O
so	O
-	O
called	O
sanroque	O
mutation	O
in	O
Roquin	B-protein
-	I-protein
1	I-protein
,	O
a	O
single	O
amino	O
acid	O
exchange	O
from	O
Met199	B-residue_name_number
to	O
Arg	B-residue_name
that	O
causes	O
the	O
development	O
of	O
systemic	O
lupus	O
erythematosus	O
-	O
like	O
symptoms	O
in	O
homozygous	O
mice	B-taxonomy_domain
.	O

The	O
Rc3h1	B-gene
and	O
Rc3h2	B-gene
genes	O
,	O
encoding	O
for	O
Roquin	B-protein
-	I-protein
1	I-protein
and	O
Roquin	B-protein
-	I-protein
2	I-protein
proteins	O
in	O
vertebrates	B-taxonomy_domain
,	O
respectively	O
,	O
have	O
both	O
been	O
shown	O
to	O
be	O
essential	O
for	O
the	O
survival	O
of	O
mice	B-taxonomy_domain
,	O
but	O
apparently	O
serve	O
redundant	O
functions	O
in	O
T	O
cells	O
.	O

Consistently	O
,	O
CD4	O
+	O
and	O
CD8	O
+	O
T	O
cells	O
with	O
the	O
combined	O
deletion	B-experimental_method
of	I-experimental_method
Roquin	B-protein
-	O
encoding	O
genes	O
are	O
spontaneously	O
activated	O
and	O
CD4	O
+	O
T	O
-	O
helper	O
cells	O
preferentially	O
differentiate	O
into	O
the	O
Th1	O
,	O
Tfh	O
or	O
Th17	O
subsets	O
.	O

Roquin	B-protein
-	I-protein
1	I-protein
was	O
shown	O
to	O
negatively	O
regulate	O
expression	O
of	O
transcripts	O
encoding	O
for	O
co	B-protein_type
-	I-protein_type
stimulatory	I-protein_type
receptors	I-protein_type
such	O
as	O
Icos	B-protein
,	O
Ox40	B-protein
and	O
CTLA	B-protein
-	I-protein
4	I-protein
,	O
for	O
cytokines	B-protein_type
such	O
as	O
interleukin	B-protein
(	I-protein
IL	I-protein
)-	I-protein
6	I-protein
and	O
tumour	B-protein
necrosis	I-protein
factor	I-protein
or	O
for	O
transcription	B-protein_type
factors	I-protein_type
such	O
as	O
IRF4	B-protein
,	O
IκBNS	B-protein
and	O
IκBζ	B-protein
(	O
refs	O
).	O

We	O
have	O
recently	O
reported	O
structural	B-evidence
and	I-evidence
functional	I-evidence
data	I-evidence
of	O
the	O
Roquin	B-protein
-	I-protein
1	I-protein
ROQ	B-structure_element
domain	O
bound	B-protein_state
to	I-protein_state
a	O
canonical	O
constitutive	B-structure_element
decay	I-structure_element
element	I-structure_element
(	O
CDE	B-structure_element
),	O
a	O
short	B-structure_element
stem	I-structure_element
loop	I-structure_element
(	O
SL	B-structure_element
)	O
that	O
acts	O
as	O
a	O
cis	O
-	O
regulatory	O
RNA	B-chemical
element	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
untranslated	I-structure_element
regions	I-structure_element
(	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
)	O
of	O
target	O
genes	O
such	O
as	O
Tnf	B-protein
(	O
ref	O
).	O

The	O
ROQ	B-structure_element
domain	O
adopts	O
an	O
extended	B-structure_element
winged	I-structure_element
helix	I-structure_element
fold	I-structure_element
that	O
engages	O
predominantly	O
non	O
-	O
sequence	O
-	O
specific	O
protein	O
–	O
RNA	B-chemical
contacts	O
and	O
mainly	O
recognizes	O
the	O
shape	O
of	O
the	O
canonical	O
Tnf	B-protein
CDE	B-structure_element
RNA	B-chemical
.	O

The	O
structural	B-evidence
data	I-evidence
and	O
mutational	B-experimental_method
analysis	I-experimental_method
indicated	O
that	O
a	O
broader	O
,	O
extended	O
range	O
of	O
sequence	O
variations	O
in	O
both	O
the	O
loop	B-structure_element
and	O
stem	B-structure_element
of	O
the	O
CDE	B-structure_element
element	O
is	O
recognized	O
and	O
regulated	O
by	O
Roquin	B-protein
.	O

At	O
the	O
same	O
time	O
,	O
Tan	O
et	O
al	O
.	O
described	O
the	O
crystal	B-evidence
structure	I-evidence
and	O
supporting	O
functional	O
data	O
of	O
a	O
similar	O
interaction	O
with	O
a	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
,	O
and	O
reported	O
a	O
second	B-site
binding	I-site
site	I-site
for	O
a	O
double	B-chemical
-	I-chemical
stranded	I-chemical
RNA	I-chemical
(	O
dsRNA	B-chemical
)	O
within	O
an	O
extended	B-protein_state
ROQ	B-structure_element
domain	O
.	O

The	O
structural	O
basis	O
for	O
CDE	B-structure_element
recognition	O
by	O
the	O
Roquin	B-protein
-	I-protein
2	I-protein
ROQ	B-structure_element
domain	O
has	O
also	O
been	O
recently	O
reported	O
.	O

We	O
found	O
that	O
the	O
posttranscriptional	O
activity	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
and	O
Roquin	B-protein
-	I-protein
2	I-protein
is	O
regulated	O
through	O
cleavage	O
by	O
the	O
paracaspase	B-protein_type
MALT1	B-protein
(	O
refs	O
).	O

Enhanced	O
MALT1	B-protein
-	O
dependent	O
cleavage	O
and	O
inactivation	O
of	O
Roquin	B-protein
,	O
and	O
thus	O
less	O
effective	O
repression	O
of	O
target	O
genes	O
,	O
result	O
from	O
increased	O
strength	O
of	O
antigen	O
recognition	O
in	O
T	O
cells	O
.	O

These	O
findings	O
suggest	O
that	O
dependent	O
on	O
the	O
strength	O
of	O
cognate	O
antigen	O
recognition	O
differential	O
gene	O
expression	O
and	O
cell	O
fate	O
decisions	O
can	O
be	O
established	O
in	O
naive	O
T	O
cells	O
by	O
a	O
graded	O
cleavage	O
and	O
inactivation	O
of	O
Roquin	B-protein
.	O

In	O
addition	O
to	O
this	O
mechanism	O
,	O
the	O
composition	O
and	O
binding	B-evidence
affinity	I-evidence
of	O
cis	O
-	O
regulatory	O
SL	B-structure_element
elements	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
of	O
target	O
mRNAs	B-chemical
may	O
determine	O
the	O
sensitivity	O
to	O
repression	O
by	O
the	O
trans	O
-	O
acting	O
factor	O
Roquin	B-protein
.	O
Defining	O
the	O
SL	B-structure_element
RNA	B-chemical
structures	O
that	O
are	O
recognized	O
by	O
Roquin	B-protein
is	O
therefore	O
essential	O
for	O
our	O
understanding	O
of	O
posttranscriptional	O
gene	O
regulation	O
by	O
Roquin	B-protein
and	O
its	O
involvement	O
in	O
T	O
-	O
cell	O
biology	O
and	O
T	O
-	O
cell	O
-	O
driven	O
pathology	O
.	O

Here	O
we	O
present	O
structural	O
and	O
functional	O
evidence	O
for	O
a	O
greatly	O
expanded	O
repertoire	O
of	O
RNA	B-chemical
elements	O
that	O
are	O
regulated	O
by	O
Roquin	B-protein
as	O
demonstrated	O
with	O
a	O
novel	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
hexaloop	I-structure_element
SL	B-structure_element
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Ox40	B-protein
bound	B-protein_state
to	I-protein_state
the	O
Roquin	B-protein
-	I-protein
1	I-protein
ROQ	B-structure_element
domain	O
.	O

We	O
find	O
an	O
additive	O
regulation	O
of	O
Ox40	B-protein
gene	O
expression	O
based	O
on	O
both	O
its	O
CDE	B-structure_element
-	O
like	O
and	O
hexaloop	B-structure_element
SL	B-structure_element
RNAs	B-chemical
that	O
we	O
identified	O
using	O
Systematic	B-experimental_method
Evolution	I-experimental_method
of	I-experimental_method
Ligands	I-experimental_method
by	I-experimental_method
Exponential	I-experimental_method
Enrichment	I-experimental_method
(	O
SELEX	B-experimental_method
)	O
experiments	O
.	O

Our	O
X	B-experimental_method
-	I-experimental_method
ray	I-experimental_method
crystallographic	I-experimental_method
,	O
NMR	B-experimental_method
,	O
biochemical	B-evidence
and	I-evidence
functional	I-evidence
data	I-evidence
combined	O
with	O
mutational	B-experimental_method
analysis	I-experimental_method
demonstrate	O
that	O
both	O
triloop	B-structure_element
and	O
hexaloop	B-structure_element
SL	B-structure_element
RNAs	B-chemical
contribute	O
to	O
the	O
functional	O
activity	O
of	O
Roquin	B-protein
in	O
T	O
cells	O
.	O

SELEX	B-experimental_method
identifies	O
novel	O
RNA	B-chemical
ligands	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein

We	O
set	O
out	O
to	O
identify	O
Roquin	B-protein_state
-	I-protein_state
bound	I-protein_state
RNA	B-chemical
motifs	O
in	O
an	O
unbiased	O
manner	O
by	O
performing	O
SELEX	B-experimental_method
experiments	O
.	O

A	O
biotinylated	B-protein_state
amino	O
-	O
terminal	O
protein	O
fragment	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
(	O
residues	O
2	B-residue_range
–	I-residue_range
440	I-residue_range
)	O
was	O
used	O
to	O
enrich	O
RNAs	B-chemical
from	O
a	O
library	O
containing	O
47	O
random	O
nucleotides	O
over	O
three	O
sequential	O
selection	O
rounds	O
.	O

Next	B-experimental_method
-	I-experimental_method
generation	I-experimental_method
sequencing	I-experimental_method
(	O
NGS	B-experimental_method
)	O
of	O
the	O
RNA	B-chemical
before	O
and	O
after	O
each	O
selection	O
round	O
revealed	O
that	O
the	O
starting	O
pool	O
represented	O
about	O
99	O
.	O
6	O
%	O
unique	O
reads	O
in	O
∼	O
4	O
.	O
2	O
×	O
106	O
sequences	O
.	O

Bioinformatic	B-experimental_method
analysis	I-experimental_method
of	O
NGS	B-experimental_method
data	O
sets	O
derived	O
from	O
the	O
starting	O
pool	O
and	O
enriched	O
selection	O
rounds	O
revealed	O
that	O
the	O
complexity	O
was	O
reduced	O
to	O
78	O
.	O
6	O
%	O
unique	O
reads	O
in	O
3	O
.	O
7	O
×	O
106	O
sequences	O
that	O
were	O
analysed	O
after	O
3	O
rounds	O
of	O
selection	O
and	O
enrichment	O
.	O

For	O
NGS	B-experimental_method
data	O
analysis	O
,	O
the	O
COMPAS	O
software	O
(	O
AptaIT	O
,	O
Munich	O
,	O
Germany	O
)	O
was	O
applied	O
.	O

Enriched	O
sequences	B-experimental_method
were	I-experimental_method
clustered	I-experimental_method
into	O
so	O
-	O
called	O
patterns	O
with	O
highly	O
homologous	O
sequences	O
.	O

Based	O
on	O
this	O
so	O
-	O
called	O
co	B-experimental_method
-	I-experimental_method
occurrence	I-experimental_method
approach	I-experimental_method
,	O
patterns	O
on	O
the	O
basis	O
of	O
frequent	O
motifs	O
were	O
generated	O
and	O
were	O
searched	O
for	O
prominent	O
hexamer	O
sequences	O
(	O
Supplementary	O
Fig	O
.	O
1a	O
).	O

We	O
identified	O
5	B-chemical
′-	I-chemical
CGTTTT	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
5	B-chemical
′-	I-chemical
GCGTTT	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
5	B-chemical
′-	I-chemical
TGCGTT	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
and	O
5	B-chemical
′-	I-chemical
GTTTTA	I-chemical
-	I-chemical
3	I-chemical
′	I-chemical
motifs	O
that	O
were	O
also	O
reconfirmed	O
in	O
an	O
independent	O
experiment	O
(	O
Supplementary	O
Fig	O
.	O
1a	O
)	O
and	O
are	O
located	O
within	O
highly	O
similar	O
sequences	O
(	O
Fig	O
.	O
1a	O
and	O
Supplementary	O
Fig	O
.	O
1b	O
).	O

Consistent	O
with	O
previous	O
findings	O
showing	O
that	O
the	O
sanroque	B-mutant
mutation	I-mutant
does	O
not	O
impair	O
RNA	B-chemical
binding	O
of	O
Roquin	B-protein
,	O
we	O
found	O
similarly	O
enriched	O
sequences	O
in	O
SELEX	B-experimental_method
approaches	O
using	O
a	O
corresponding	O
Roquin	B-protein
-	I-protein
1	I-protein
fragment	O
harbouring	O
the	O
M199R	B-mutant
mutation	O
(	O
Fig	O
.	O
1a	O
and	O
Supplementary	O
Fig	O
.	O
1b	O
).	O

Notably	O
,	O
our	O
SELEX	B-experimental_method
approach	O
did	O
not	O
reveal	O
the	O
previously	O
identified	O
CDE	B-structure_element
sequence	O
.	O

We	O
assume	O
that	O
the	O
region	O
of	O
sequence	O
identity	O
in	O
the	O
CDE	B-structure_element
is	O
too	O
short	O
for	O
our	O
sequence	B-experimental_method
clustering	I-experimental_method
algorithm	I-experimental_method
.	O

Evaluation	O
of	O
the	O
structural	O
context	O
for	O
the	O
SELEX	B-experimental_method
-	O
derived	O
motif	O
suggested	O
a	O
putative	O
SL	B-structure_element
formation	O
with	O
six	O
unpaired	O
nucleotides	O
in	O
a	O
loop	B-structure_element
followed	O
by	O
a	O
5	O
–	O
8	O
nt	O
stem	B-structure_element
,	O
with	O
one	O
base	O
in	O
the	O
stem	B-structure_element
not	O
being	O
paired	O
(	O
Supplementary	O
Fig	O
.	O
1c	O
).	O

Searching	O
the	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
of	O
known	O
Roquin	B-protein
targets	O
with	O
the	O
consensus	O
5	B-chemical
′-	I-chemical
TGCGTTTTAGGA	I-chemical
-	I-chemical
3	I-chemical
′,	I-chemical
obtained	O
by	O
Motif	B-experimental_method
-	I-experimental_method
based	I-experimental_method
sequence	I-experimental_method
analysis	I-experimental_method
(	O
MEME	B-experimental_method
),	O
revealed	O
a	O
homologous	O
sequence	O
with	O
the	O
potential	O
to	O
form	O
a	O
hexaloop	B-structure_element
structure	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Ox40	B-protein
(	O
Fig	O
.	O
1b	O
).	O

Importantly	O
,	O
this	O
motif	O
is	O
present	O
across	O
species	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
of	O
respective	O
mRNAs	B-chemical
and	O
showed	O
highest	O
conservation	O
in	O
the	O
loop	B-structure_element
and	O
the	O
upper	O
stem	B-structure_element
sequences	O
with	O
a	O
drop	O
of	O
conservation	O
towards	O
the	O
boundaries	O
of	O
the	O
motif	O
(	O
Fig	O
.	O
1c	O
,	O
d	O
).	O

The	O
predicted	O
SL	B-structure_element
for	O
the	O
consensus	O
SELEX	B-experimental_method
-	O
derived	O
motif	O
(	O
from	O
here	O
on	O
referred	O
to	O
as	O
alternative	B-structure_element
decay	I-structure_element
element	I-structure_element
SL	B-structure_element
,	O
ADE	B-structure_element
SL	B-structure_element
),	O
the	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
,	O
is	O
positioned	O
5	O
′	O
to	O
another	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Ox40	B-protein
mRNA	B-chemical
.	O

This	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
differs	O
in	O
the	O
sequence	O
of	O
the	O
upper	O
stem	O
from	O
the	O
canonical	O
CDE	B-structure_element
from	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Tnf	B-protein
mRNA	B-chemical
(	O
CDE	B-structure_element
SL	B-structure_element
)	O
(	O
Fig	O
.	O
1d	O
).	O

NMR	B-experimental_method
analysis	O
of	O
Roquin	B-protein_state
-	I-protein_state
bound	I-protein_state
SL	B-structure_element
RNAs	B-chemical

We	O
used	O
NMR	B-experimental_method
to	O
analyse	O
the	O
secondary	O
structure	O
of	O
Roquin	B-structure_element
-	I-structure_element
1	I-structure_element
-	I-structure_element
binding	I-structure_element
motifs	I-structure_element
derived	O
from	O
SELEX	B-experimental_method
.	O

Imino	B-experimental_method
one	I-experimental_method
-	I-experimental_method
and	I-experimental_method
two	I-experimental_method
-	I-experimental_method
dimensional	I-experimental_method
nuclear	I-experimental_method
Overhauser	I-experimental_method
enhancement	I-experimental_method
spectroscopy	I-experimental_method
(	O
NOESY	B-experimental_method
)	O
NMR	B-experimental_method
spectra	B-evidence
of	O
the	O
free	B-protein_state
RNA	B-chemical
and	O
when	O
bound	B-protein_state
to	I-protein_state
the	O
Roquin	B-protein
-	I-protein
1	I-protein
ROQ	B-structure_element
domain	O
were	O
recorded	O
for	O
the	O
ADE	B-structure_element
SL	B-structure_element
,	O
the	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Ox40	B-protein
and	O
the	O
previously	O
identified	O
Ox40	B-protein
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
Fig	O
.	O
2	O
).	O

The	O
NMR	B-experimental_method
data	O
of	O
the	O
free	B-protein_state
RNAs	B-chemical
show	O
that	O
almost	O
all	O
predicted	O
base	O
pairs	O
in	O
the	O
stem	B-structure_element
regions	I-structure_element
of	O
the	O
hexa	B-structure_element
-	I-structure_element
and	I-structure_element
triloop	I-structure_element
SL	B-structure_element
including	O
the	O
closing	O
base	O
pairs	O
are	O
formed	O
in	O
all	O
three	O
RNAs	B-chemical
.	O

Notably	O
,	O
we	O
also	O
found	O
an	O
unambiguous	O
imino	O
proton	O
signal	O
for	O
G15	B-residue_name_number
,	O
but	O
not	O
G6	B-residue_name_number
,	O
in	O
the	O
ADE	B-structure_element
SL	B-structure_element
,	O
indicating	O
a	O
non	B-bond_interaction
-	I-bond_interaction
Watson	I-bond_interaction
–	I-bond_interaction
Crick	I-bond_interaction
G	I-bond_interaction
–	I-bond_interaction
G	I-bond_interaction
base	I-bond_interaction
pair	I-bond_interaction
at	O
this	O
position	O
(	O
Fig	O
.	O
2a	O
).	O

Significant	O
chemical	B-evidence
shift	I-evidence
perturbations	I-evidence
(	O
CSPs	B-evidence
)	O
are	O
observed	O
for	O
imino	O
proton	O
signals	O
on	O
binding	O
to	O
the	O
ROQ	B-structure_element
domain	O
,	O
demonstrating	O
that	O
formation	O
of	O
protein	O
–	O
RNA	B-chemical
complexes	O
involves	O
contacts	O
of	O
the	O
ROQ	B-structure_element
domain	O
to	O
the	O
stem	B-structure_element
region	I-structure_element
of	O
the	O
RNA	B-chemical
ligands	O
(	O
Fig	O
.	O
2	O
,	O
bases	O
coloured	O
red	O
).	O

No	O
imino	O
correlations	O
are	O
observed	O
for	O
the	O
predicted	O
Watson	B-bond_interaction
–	I-bond_interaction
Crick	I-bond_interaction
base	I-bond_interaction
pairs	I-bond_interaction
at	O
the	O
bottom	O
of	O
the	O
ADE	B-structure_element
SL	B-structure_element
and	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
,	O
as	O
well	O
as	O
for	O
the	O
A	B-residue_name
–	O
U	B-residue_name
base	O
pair	O
flanking	O
the	O
bulge	B-structure_element
in	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
(	O
Fig	O
.	O
2a	O
,	O
b	O
),	O
suggesting	O
that	O
these	O
base	O
pairs	O
are	O
dynamic	O
.	O

In	O
contrast	O
,	O
all	O
expected	O
base	O
pairs	O
are	O
observed	O
for	O
the	O
Ox40	B-protein
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
(	O
Fig	O
.	O
2c	O
;	O
see	O
also	O
Supplementary	O
Notes	O
).	O

Structures	B-evidence
of	O
ROQ	B-structure_element
bound	B-protein_state
to	I-protein_state
ADE	B-structure_element
SL	B-structure_element
RNAs	B-chemical

To	O
elucidate	O
how	O
Roquin	B-protein
can	O
recognize	O
the	O
novel	O
SL	B-structure_element
elements	O
identified	O
in	O
the	O
SELEX	B-experimental_method
approach	O
,	O
we	O
solved	B-experimental_method
crystal	B-evidence
structures	I-evidence
of	O
the	O
Roquin	B-protein
-	I-protein
1	I-protein
ROQ	B-structure_element
domain	O
bound	B-protein_state
to	I-protein_state
these	O
non	O
-	O
canonical	O
RNA	B-chemical
elements	O
.	O

The	O
structures	B-evidence
of	O
ROQ	B-structure_element
bound	B-protein_state
to	I-protein_state
the	O
20	O
-	O
mer	O
ADE	B-structure_element
SL	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
2a	O
)	O
and	O
to	O
the	O
22	O
-	O
mer	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
(	O
Fig	O
.	O
3a	O
)	O
were	O
refined	O
to	O
a	O
resolution	O
of	O
3	O
.	O
0	O
and	O
2	O
.	O
2	O
Å	O
,	O
respectively	O
.	O

In	O
both	O
structures	B-evidence
the	O
RNA	B-chemical
adopts	O
an	O
SL	B-structure_element
fold	O
,	O
where	O
the	O
hexaloop	B-structure_element
is	O
located	O
in	O
the	O
vicinity	O
of	O
the	O
carboxy	O
-	O
terminal	O
end	O
of	O
ROQ	B-structure_element
helix	B-structure_element
α4	B-structure_element
and	O
the	O
N	O
-	O
terminal	O
part	O
of	O
β3	B-structure_element
(	O
Fig	O
.	O
3a	O
,	O
b	O
and	O
Supplementary	O
Fig	O
.	O
2a	O
,	O
b	O
).	O

The	O
dsRNA	B-chemical
stem	B-structure_element
is	O
recognized	O
in	O
the	O
same	O
way	O
as	O
previously	O
reported	O
for	O
the	O
Tnf	B-protein
CDE	B-structure_element
SL	B-structure_element
RNA	B-chemical
(	O
Supplementary	O
Fig	O
.	O
2c	O
–	O
e	O
).	O

As	O
may	O
be	O
expected	O
,	O
the	O
recognition	O
of	O
the	O
hexaloop	B-structure_element
is	O
significantly	O
different	O
from	O
the	O
triloop	B-structure_element
in	O
the	O
CDE	B-structure_element
RNA	B-chemical
(	O
Fig	O
.	O
3b	O
,	O
c	O
and	O
Supplementary	O
Fig	O
.	O
2b	O
).	O

Interestingly	O
,	O
although	O
the	O
sequences	O
of	O
the	O
ADE	B-structure_element
SL	B-structure_element
and	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
are	O
different	O
,	O
the	O
overall	O
structures	B-evidence
and	O
protein	O
–	O
RNA	B-chemical
contacts	O
are	O
virtually	O
identical	O
(	O
Supplementary	O
Fig	O
.	O
2a	O
,	O
d	O
,	O
e	O
).	O

The	O
only	O
differences	O
are	O
a	O
C19	B-residue_name_number
bulge	B-structure_element
,	O
the	O
non	B-bond_interaction
-	I-bond_interaction
Watson	I-bond_interaction
–	I-bond_interaction
Crick	I-bond_interaction
G6	B-residue_name_number
–	O
G15	B-residue_name_number
base	B-bond_interaction
pair	I-bond_interaction
and	O
the	O
interaction	O
of	O
U1	B-residue_name_number
with	O
Trp184	B-residue_name_number
and	O
Phe194	B-residue_name_number
in	O
the	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
(	O
Supplementary	O
Fig	O
.	O
2a	O
,	O
e	O
–	O
g	O
).	O

Given	O
their	O
highly	O
similar	O
binding	O
modes	O
we	O
focus	O
the	O
following	O
discussion	O
on	O
the	O
structure	B-evidence
of	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
,	O
as	O
it	O
naturally	O
exists	O
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
and	O
was	O
solved	O
at	O
higher	O
resolution	O
.	O

The	O
overall	O
orientation	O
and	O
recognition	O
of	O
the	O
double	B-structure_element
-	I-structure_element
stranded	I-structure_element
stem	I-structure_element
in	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
is	O
similar	O
to	O
the	O
CDE	B-structure_element
triloop	B-structure_element
.	O

Notably	O
,	O
the	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
hexaloop	I-structure_element
in	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
binds	O
to	O
an	O
extended	O
surface	B-site
on	O
the	O
ROQ	B-structure_element
domain	O
that	O
cannot	O
be	O
accessed	O
by	O
the	O
CDE	B-structure_element
triloop	B-structure_element
(	O
Fig	O
.	O
3b	O
,	O
c	O
)	O
and	O
includes	O
a	O
few	O
pyrimidine	O
-	O
specific	O
contacts	O
.	O

For	O
example	O
,	O
the	O
main	O
chain	O
atoms	O
of	O
Phe255	B-residue_name_number
form	O
two	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
the	O
Watson	O
–	O
Crick	O
face	O
of	O
the	O
U11	B-residue_name_number
base	O
(	O
Fig	O
.	O
3d	O
).	O

Although	O
in	O
the	O
structure	B-evidence
of	O
the	O
Tnf	B-protein
CDE	B-structure_element
triloop	B-structure_element
the	O
Tyr250	B-residue_name_number
side	O
chain	O
engages	O
only	O
one	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
to	O
the	O
phosphate	O
group	O
of	O
G12	B-residue_name_number
(	O
ref	O
.),	O
a	O
number	O
of	O
contacts	O
are	O
observed	O
with	O
the	O
hexaloop	B-structure_element
(	O
Fig	O
.	O
3d	O
–	O
f	O
):	O
the	O
side	O
chain	O
hydroxyl	O
of	O
Tyr250	B-residue_name_number
contacts	O
the	O
phosphate	O
group	O
of	O
U11	B-residue_name_number
,	O
while	O
the	O
aromatic	O
ring	O
is	O
positioned	O
by	O
parallel	O
and	O
orthogonal	O
stacking	B-bond_interaction
interactions	I-bond_interaction
with	O
the	O
U10	B-residue_name_number
and	O
U11	B-residue_name_number
bases	O
,	O
on	O
either	O
side	O
,	O
respectively	O
(	O
Fig	O
.	O
3e	O
).	O

In	O
addition	O
,	O
the	O
Tyr250	B-residue_name_number
main	O
-	O
chain	O
carbonyl	O
interacts	O
with	O
U13	B-residue_name_number
imino	O
proton	O
(	O
Fig	O
.	O
3d	O
,	O
e	O
).	O

Val257	B-residue_name_number
and	O
Lys259	B-residue_name_number
in	O
strand	B-structure_element
β3	B-structure_element
are	O
too	O
far	O
to	O
contact	O
the	O
UGU	B-structure_element
triloop	B-structure_element
in	O
the	O
Tnf	B-protein
CDE	B-structure_element
RNA	B-chemical
,	O
but	O
mediate	O
a	O
number	O
of	O
contacts	O
with	O
the	O
longer	O
hexaloop	B-structure_element
.	O

The	O
side	O
chain	O
of	O
Lys259	B-residue_name_number
forms	O
hydrogen	B-bond_interaction
bonds	I-bond_interaction
with	O
the	O
phosphate	O
groups	O
of	O
U10	B-residue_name_number
and	O
U11	B-residue_name_number
(	O
Fig	O
.	O
3e	O
,	O
f	O
)	O
and	O
the	O
hydrophobic	O
side	O
chain	O
of	O
Val257	B-residue_name_number
stacks	B-bond_interaction
with	O
the	O
U11	B-residue_name_number
base	O
(	O
Fig	O
.	O
3d	O
,	O
f	O
).	O

The	O
RNA	B-chemical
stem	B-structure_element
is	O
closed	O
by	O
a	O
Watson	B-bond_interaction
–	I-bond_interaction
Crick	I-bond_interaction
base	I-bond_interaction
pair	I-bond_interaction
(	O
C8	B-residue_name_number
–	O
G15	B-residue_name_number
in	O
the	O
hexaloop	B-structure_element
SL	B-structure_element
RNA	B-chemical
).	O

Interestingly	O
,	O
the	O
G9	B-residue_name_number
base	O
stacks	B-bond_interaction
on	O
top	O
of	O
this	O
closing	O
base	O
pair	O
and	O
takes	O
a	O
position	O
that	O
is	O
very	O
similar	O
to	O
the	O
purine	O
base	O
of	O
G12	B-residue_name_number
in	O
the	O
CDE	B-structure_element
triloop	B-structure_element
(	O
Fig	O
.	O
3b	O
,	O
c	O
and	O
Supplementary	O
Fig	O
.	O
2b	O
).	O

The	O
G9	B-residue_name_number
base	O
does	O
not	O
form	O
a	O
base	O
pair	O
with	O
A14	B-residue_name_number
but	O
rather	O
the	O
A14	B-residue_name_number
base	O
packs	O
into	O
the	O
minor	B-site
groove	I-site
of	O
the	O
RNA	B-chemical
duplex	O
.	O

This	O
arrangement	O
provides	O
an	O
extended	O
stacking	B-bond_interaction
interaction	I-bond_interaction
of	O
G9	B-residue_name_number
,	O
U10	B-residue_name_number
and	O
Tyr250	B-residue_name_number
in	O
the	O
ROQ	B-structure_element
domain	O
at	O
the	O
5	O
′-	O
side	O
of	O
the	O
RNA	B-chemical
stem	B-structure_element
(	O
Fig	O
.	O
3e	O
).	O

The	O
U11	B-residue_name_number
and	O
U13	B-residue_name_number
bases	O
stack	B-bond_interaction
with	O
each	O
other	O
in	O
the	O
vicinity	O
of	O
the	O
ROQ	B-structure_element
domain	O
wing	B-structure_element
(	O
Fig	O
.	O
3b	O
,	O
d	O
,	O
f	O
).	O

This	O
is	O
possible	O
by	O
exposing	O
the	O
base	O
C12	B-residue_name_number
of	O
the	O
Ox	B-protein
-	I-protein
40	I-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
towards	O
the	O
solvent	O
,	O
which	O
accordingly	O
does	O
not	O
show	O
any	O
contacts	O
to	O
the	O
protein	O
.	O

In	O
summary	O
,	O
similar	O
to	O
the	O
CDE	B-structure_element
SL	B-structure_element
,	O
both	O
the	O
ADE	B-structure_element
SL	B-structure_element
and	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
are	O
recognized	O
mainly	O
by	O
non	O
-	O
sequence	O
-	O
specific	O
contacts	O
.	O

However	O
,	O
these	O
involve	O
an	O
extended	O
binding	O
surface	O
on	O
the	O
ROQ	B-structure_element
domain	O
with	O
a	O
number	O
of	O
additional	O
residues	O
compared	O
with	O
the	O
triloop	O
RNA	B-chemical
.	O

NMR	B-experimental_method
analysis	O
of	O
ROQ	B-structure_element
interactions	O
with	O
ADE	B-structure_element
SLs	B-structure_element

We	O
next	O
used	O
NMR	B-experimental_method
spectroscopy	I-experimental_method
to	O
compare	O
the	O
ROQ	B-structure_element
domain	O
interaction	O
of	O
ADE	B-structure_element
-	O
like	O
and	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
in	O
solution	O
.	O

CSPs	B-evidence
observed	O
for	O
amides	O
in	O
the	O
ROQ	B-structure_element
domain	O
on	O
binding	O
to	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
(	O
Fig	O
.	O
4a	O
,	O
b	O
)	O
map	O
to	O
residues	O
that	O
also	O
mediate	O
key	O
interactions	O
with	O
CDE	B-structure_element
SLs	B-structure_element
,	O
such	O
as	O
Lys220	B-residue_name_number
,	O
Lys239	B-residue_name_number
/	O
Thr240	B-residue_name_number
and	O
Lys259	B-residue_name_number
/	O
Arg260	B-residue_name_number
(	O
Fig	O
.	O
4b	O
).	O

This	O
is	O
fully	O
consistent	O
with	O
the	O
interactions	O
observed	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
(	O
Supplementary	O
Fig	O
.	O
2c	O
–	O
e	O
)	O
and	O
indicates	O
a	O
similar	O
binding	B-site
surface	I-site
.	O

However	O
,	O
there	O
are	O
also	O
notable	O
CSP	B-evidence
differences	I-evidence
when	O
comparing	O
binding	O
of	O
the	O
ROQ	B-structure_element
domain	O
to	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
and	O
to	O
the	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
(	O
Fig	O
.	O
4c	O
),	O
or	O
to	O
the	O
Tnf	B-protein
CDE	B-structure_element
SL	B-structure_element
RNA	B-chemical
(	O
Supplementary	O
Fig	O
.	O
3	O
and	O
Supplementary	O
Notes	O
).	O

For	O
example	O
,	O
Ser253	B-residue_name_number
is	O
strongly	O
affected	O
only	O
on	O
binding	O
to	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
Fig	O
.	O
4a	O
,	O
b	O
)	O
in	O
line	O
with	O
tight	O
interactions	O
with	O
the	O
hexaloop	B-structure_element
(	O
Fig	O
.	O
3d	O
).	O

On	O
the	O
other	O
hand	O
,	O
comparison	O
of	O
ROQ	B-structure_element
domain	O
binding	O
with	O
the	O
ADE	B-structure_element
and	O
with	O
the	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
indicates	O
almost	O
identical	O
NMR	B-experimental_method
spectra	B-evidence
and	O
CSPs	B-evidence
.	O

This	O
is	O
consistent	O
with	O
the	O
very	O
similar	O
structural	O
features	O
and	O
mode	O
of	O
RNA	B-chemical
recognition	O
of	O
the	O
ROQ	B-structure_element
domain	O
with	O
these	O
RNAs	B-chemical
(	O
Supplementary	O
Fig	O
.	O
2a	O
,	O
d	O
,	O
e	O
).	O

Mutational	B-experimental_method
analysis	I-experimental_method
of	O
the	O
ROQ	B-structure_element
-	O
ADE	B-structure_element
interaction	O

To	O
examine	O
the	O
individual	O
contributions	O
of	O
ROQ	B-structure_element
–	O
hexaloop	O
interactions	O
for	O
complex	O
formation	O
,	O
we	O
performed	O
electrophoretic	B-experimental_method
mobility	I-experimental_method
shift	I-experimental_method
assays	I-experimental_method
(	O
EMSAs	B-experimental_method
)	O
with	O
variants	O
of	O
the	O
ROQ	B-structure_element
domain	O
and	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
RNA	B-chemical
(	O
Fig	O
.	O
5a	O
and	O
Supplementary	O
Fig	O
.	O
4	O
).	O

Analysis	O
of	O
the	O
interaction	O
with	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
ROQ	B-structure_element
revealed	O
an	O
apparent	O
affinity	B-evidence
in	O
a	O
similar	O
range	O
as	O
for	O
the	O
Tnf	B-protein
CDE	B-structure_element
(	O
Fig	O
.	O
5a	O
and	O
)	O
Table	O
2	O
).	O

We	O
next	O
tested	O
a	O
set	O
of	O
mutants	O
(	O
Supplementary	O
Fig	O
.	O
4	O
),	O
which	O
were	O
designed	O
based	O
on	O
contacts	O
observed	O
in	O
the	O
crystal	B-evidence
structure	I-evidence
(	O
Fig	O
.	O
3	O
)	O
and	O
the	O
NMR	B-experimental_method
CSPs	B-evidence
(	O
Fig	O
.	O
4a	O
,	O
b	O
).	O

In	O
line	O
with	O
expectations	O
from	O
ROQ	B-complex_assembly
-	I-complex_assembly
Tnf	I-complex_assembly
CDE	I-complex_assembly
binding	O
(	O
see	O
comparison	O
in	O
Supplementary	O
Fig	O
.	O
4	O
)	O
and	O
based	O
on	O
our	O
structural	B-experimental_method
analysis	I-experimental_method
,	O
the	O
key	O
residues	O
Lys220	B-residue_name_number
,	O
Lys239	B-residue_name_number
,	O
Lys259	B-residue_name_number
and	O
Arg260	B-residue_name_number
strongly	O
reduce	O
or	O
abolish	O
binding	O
after	O
replacement	B-experimental_method
by	O
alanine	B-residue_name
.	O

We	O
also	O
observe	O
an	O
almost	O
complete	O
loss	O
of	O
binding	O
in	O
the	O
Y250A	B-mutant
mutant	B-protein_state
to	O
the	O
hexaloop	B-structure_element
SL	B-structure_element
RNA	B-chemical
,	O
which	O
had	O
not	O
been	O
seen	O
for	O
the	O
Tnf	B-protein
CDE	B-structure_element
previously	O
(	O
Fig	O
.	O
5a	O
).	O

This	O
underlines	O
the	O
central	O
role	O
of	O
Tyr250	B-residue_name_number
for	O
stabilization	O
of	O
the	O
hexaloop	B-structure_element
structure	O
and	O
recognition	O
by	O
stacking	B-bond_interaction
interactions	I-bond_interaction
(	O
Fig	O
.	O
3b	O
,	O
e	O
).	O

Mutation	B-experimental_method
of	O
Ser253	B-residue_name_number
,	O
which	O
shows	O
large	O
CSPs	B-evidence
in	O
the	O
NMR	B-experimental_method
titrations	I-experimental_method
(	O
Fig	O
.	O
4a	O
,	O
b	O
),	O
does	O
not	O
significantly	O
impair	O
complex	O
formation	O
(	O
Supplementary	O
Fig	O
.	O
4	O
).	O

The	O
large	O
chemical	B-evidence
shift	I-evidence
change	I-evidence
is	O
probably	O
caused	O
by	O
ring	O
current	O
effects	O
induced	O
by	O
the	O
close	O
proximity	O
of	O
the	O
U11	B-residue_name_number
and	O
U13	B-residue_name_number
bases	O
.	O

Finally	O
,	O
a	O
mutant	B-protein_state
in	O
the	O
wing	B-structure_element
of	O
the	O
ROQ	B-structure_element
domain	O
(	O
S265Y	B-mutant
)	O
does	O
only	O
slightly	O
impair	O
binding	O
,	O
as	O
has	O
been	O
previously	O
observed	O
for	O
the	O
interaction	O
with	O
the	O
Tnf	B-protein
CDE	B-structure_element
(	O
Supplementary	O
Fig	O
.	O
4	O
).	O

This	O
indicates	O
that	O
replacement	B-experimental_method
by	O
Tyr	B-residue_name
does	O
not	O
strongly	O
affect	O
the	O
RNA	B-chemical
interaction	O
,	O
and	O
that	O
some	O
conformational	O
variations	O
are	O
tolerated	O
.	O

Thus	O
,	O
the	O
mutational	B-experimental_method
analysis	I-experimental_method
is	O
fully	O
consistent	O
with	O
the	O
recognition	O
of	O
the	O
hexaloop	B-structure_element
observed	O
in	O
our	O
crystal	B-evidence
structures	I-evidence
.	O

To	O
prove	O
the	O
contribution	O
of	O
the	O
key	O
residue	O
Tyr250	B-residue_name_number
in	O
Roquin	B-protein
-	I-protein
1	I-protein
to	O
Ox40	B-protein
mRNA	B-chemical
recognition	O
and	O
regulation	O
,	O
we	O
set	O
up	O
a	O
retroviral	B-experimental_method
reconstitution	I-experimental_method
system	I-experimental_method
in	O
Roquin	B-protein
-	O
deficient	O
CD4	O
+	O
T	O
cells	O
.	O

Isolated	O
CD4	O
+	O
T	O
cells	O
from	O
Rc3h1	B-gene
/	O
2fl	B-gene
/	O
fl	B-gene
;	O
Cd4	O
-	O
Cre	O
-	O
ERT2	O
;	O
rtTA	O
mice	B-taxonomy_domain
harbouring	O
floxed	O
Roquin	B-protein
-	I-protein
1	I-protein
/	O
2	B-protein
encoding	O
alleles	O
,	O
a	O
tamoxifen	B-chemical
-	O
inducible	O
Cre	O
recombinase	O
and	O
the	O
reverse	B-protein_type
tetracycline	I-protein_type
-	I-protein_type
controlled	I-protein_type
transactivator	I-protein_type
rtTA	B-protein
were	O
treated	O
in	O
vitro	O
with	O
4	B-chemical
-	I-chemical
hydroxy	I-chemical
tamoxifen	I-chemical
,	O
to	O
induce	O
deletion	O
.	O

The	O
cells	O
were	O
then	O
transduced	O
with	O
doxycycline	B-chemical
-	O
inducible	O
retroviral	O
vectors	O
to	O
reconstitute	O
Roquin	B-protein
-	I-protein
1	I-protein
expression	O
(	O
Fig	O
.	O
5b	O
).	O

Depletion	O
of	O
Roquin	B-protein
proteins	O
on	O
tamoxifen	B-chemical
treatment	O
(	O
Supplementary	O
Fig	O
.	O
5a	O
)	O
strongly	O
increased	O
surface	O
expression	O
of	O
Ox40	B-protein
and	O
Icos	B-protein
(	O
Fig	O
.	O
5c	O
).	O

This	O
increase	O
in	O
surface	O
expression	O
of	O
both	O
costimulatory	B-protein_type
receptors	I-protein_type
was	O
partially	O
corrected	O
by	O
the	O
doxycycline	B-chemical
-	O
induced	O
reconstitution	O
with	O
Roquin	B-protein
-	I-protein
1	I-protein
WT	B-protein_state
protein	O
(	O
Fig	O
.	O
5c	O
left	O
panels	O
).	O

Importantly	O
,	O
no	O
effect	O
was	O
observed	O
on	O
expression	O
of	O
the	O
Y250A	B-mutant
mutant	B-protein_state
of	O
Roquin	B-protein
-	I-protein
1	I-protein
or	O
the	O
K220A	B-mutant
,	O
K239A	B-mutant
and	O
R260	B-mutant
mutant	B-protein_state
,	O
which	O
is	O
strongly	O
impaired	O
in	O
CDE	B-structure_element
SL	B-structure_element
interactions	O
(	O
Fig	O
.	O
5c	O
middle	O
and	O
right	O
panels	O
).	O

However	O
,	O
it	O
is	O
also	O
possible	O
that	O
continuous	O
overexpression	B-experimental_method
of	O
targets	O
following	O
Roquin	B-protein
deletion	O
induces	O
a	O
hyperactivated	O
state	O
in	O
the	O
T	O
cells	O
.	O

This	O
hyperactivation	O
,	O
compared	O
with	O
the	O
actual	O
posttranscriptional	O
derepression	O
,	O
may	O
contribute	O
even	O
stronger	O
to	O
the	O
increased	O
Icos	B-protein
and	O
Ox40	B-protein
expression	O
levels	O
.	O

Hence	O
,	O
our	O
structure	B-experimental_method
–	I-experimental_method
function	I-experimental_method
analyses	I-experimental_method
conclusively	O
show	O
that	O
the	O
Y250	B-residue_name_number
residue	O
is	O
essential	O
for	O
Roquin	B-protein
interaction	O
and	O
regulation	O
of	O
Ox40	B-protein
,	O
and	O
potentially	O
also	O
for	O
other	O
Roquin	B-protein
targets	O
such	O
as	O
Icos	B-protein
.	O

We	O
also	O
investigated	O
the	O
role	O
of	O
individual	O
nucleotides	O
in	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
for	O
complex	O
formation	O
with	O
the	O
ROQ	B-structure_element
domain	O
.	O

We	O
designed	O
four	O
mutants	O
(	O
Mut1	O
–	O
4	O
,	O
see	O
Supplementary	O
Fig	O
.	O
6	O
)	O
that	O
were	O
expected	O
to	O
disrupt	O
key	O
interactions	O
with	O
the	O
protein	O
according	O
to	O
our	O
co	B-evidence
-	I-evidence
crystal	I-evidence
structure	I-evidence
(	O
Fig	O
.	O
3d	O
–	O
f	O
and	O
Supplementary	O
Fig	O
.	O
2	O
).	O

NMR	B-experimental_method
analysis	O
confirmed	O
that	O
all	O
mutant	B-protein_state
RNAs	B-chemical
formed	O
the	O
same	O
base	O
pairs	O
in	O
the	O
stem	B-structure_element
region	I-structure_element
,	O
identical	O
to	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
Fig	O
.	O
2b	O
and	O
Supplementary	O
Fig	O
.	O
6	O
).	O

We	O
next	O
used	O
surface	B-experimental_method
plasmon	I-experimental_method
resonance	I-experimental_method
experiments	O
to	O
determine	O
dissociation	B-evidence
constants	I-evidence
for	O
the	O
ROQ	B-structure_element
-	O
RNA	B-chemical
interaction	O
(	O
Table	O
2	O
and	O
Supplementary	O
Fig	O
.	O
7	O
).	O

Although	O
the	O
replacement	B-experimental_method
of	O
a	O
C8	B-residue_name_number
–	O
G15	B-residue_name_number
closing	O
base	O
pair	O
by	O
A	B-residue_name
-	O
U	B-residue_name
(	O
Mut	B-mutant
4	I-mutant
)	O
only	O
reduces	O
the	O
affinity	B-evidence
threefold	O
,	O
reduction	O
of	O
loop	B-structure_element
size	O
in	O
the	O
A14C	B-mutant
mutant	B-protein_state
(	O
Mut	B-mutant
1	I-mutant
,	O
see	O
Table	O
2	O
)	O
reduces	O
the	O
affinity	B-evidence
and	O
binding	O
is	O
not	O
detected	O
by	O
surface	B-experimental_method
plasmon	I-experimental_method
resonance	I-experimental_method
.	O

As	O
intended	O
,	O
the	O
mutation	O
Mut	B-mutant
1	I-mutant
allows	O
the	O
formation	O
of	O
an	O
additional	O
base	O
pair	O
and	O
thus	O
leads	O
to	O
the	O
formation	O
of	O
a	O
tetraloop	B-structure_element
with	O
a	O
new	O
G	B-residue_name
-	O
C	B-residue_name
closing	O
base	O
pair	O
(	O
Supplementary	O
Fig	O
.	O
6a	O
).	O

Consistent	O
with	O
the	O
structural	B-experimental_method
analysis	I-experimental_method
,	O
we	O
assume	O
that	O
this	O
variant	O
alters	O
the	O
hexaloop	B-structure_element
conformation	O
and	O
thus	O
reduces	O
the	O
interaction	O
with	O
ROQ	B-structure_element
.	O

Disruption	O
of	O
stacking	B-bond_interaction
interactions	I-bond_interaction
between	O
G15	B-residue_name_number
,	O
G9	B-residue_name_number
and	O
Y250	B-residue_name_number
in	O
the	O
G9C	B-mutant
mutant	B-protein_state
(	O
Mut	B-mutant
2	I-mutant
)	O
completely	O
abolished	O
binding	O
of	O
ROQ	B-structure_element
to	O
the	O
SL	B-structure_element
RNA	B-chemical
(	O
Table	O
2	O
and	O
Supplementary	O
Fig	O
.	O
7	O
).	O

No	O
binding	O
is	O
also	O
observed	O
for	O
the	O
U11AU13G	B-mutant
double	B-protein_state
mutant	I-protein_state
(	O
Mut	B-mutant
3	I-mutant
)	O
(	O
Table	O
2	O
and	O
Supplementary	O
Fig	O
.	O
7	O
),	O
which	O
abolishes	O
specific	O
interactions	O
mediated	O
by	O
U11	B-residue_name_number
and	O
U13	B-residue_name_number
in	O
the	O
hexaloop	B-structure_element
with	O
ROQ	B-structure_element
(	O
Fig	O
.	O
3d	O
).	O

Consistent	O
with	O
the	O
SELEX	B-experimental_method
consensus	O
(	O
Fig	O
.	O
1b	O
),	O
all	O
of	O
the	O
tested	O
mutations	B-experimental_method
of	O
conserved	B-protein_state
nucleotides	B-chemical
in	O
the	O
loop	B-structure_element
reduce	O
or	O
abolish	O
the	O
interaction	O
with	O
ROQ	B-structure_element
.	O

Interestingly	O
,	O
the	O
affinity	B-evidence
of	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
Tnf	B-protein
CDE	B-structure_element
and	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SLs	B-structure_element
to	O
ROQ	B-structure_element
are	O
very	O
similar	O
(	O
42	O
and	O
81	O
nM	O
,	O
respectively	O
,	O
Table	O
2	O
and	O
Supplementary	O
Fig	O
.	O
7	O
).	O

Roquin	B-protein
binding	O
to	O
different	O
SLs	B-structure_element
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element

We	O
have	O
recently	O
shown	O
that	O
Roquin	B-protein
-	I-protein
1	I-protein
binds	O
to	O
a	O
CDE	B-structure_element
-	O
like	O
motif	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Ox40	B-protein
mRNA	B-chemical
(	O
Figs	O
1d	O
and	O
4c	O
).	O

We	O
therefore	O
investigated	O
whether	O
the	O
interactions	O
with	O
the	O
CDE	B-structure_element
-	O
like	O
and	O
the	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
both	O
contribute	O
to	O
Roquin	B-protein
-	I-protein
1	I-protein
binding	O
in	O
the	O
context	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
.	O

The	O
binding	B-evidence
affinities	I-evidence
of	O
either	O
motif	O
for	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
domain	I-structure_element
of	O
Roquin	B-protein
-	I-protein
1	I-protein
(	O
residues	O
2	B-residue_range
–	I-residue_range
440	I-residue_range
)	O
(	O
Supplementary	O
Fig	O
.	O
8a	O
,	O
b	O
)	O
or	O
the	O
ROQ	B-structure_element
domain	O
alone	B-protein_state
are	O
in	O
a	O
similar	O
range	O
(	O
Table	O
2	O
).	O

The	O
dissociation	B-evidence
constants	I-evidence
for	O
the	O
ROQ	B-structure_element
interaction	O
with	O
the	O
Ox40	B-protein
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
and	O
the	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
are	O
1	O
,	O
460	O
and	O
81	O
nM	O
,	O
respectively	O
(	O
Table	O
2	O
).	O

This	O
is	O
consistent	O
with	O
the	O
extended	O
binding	B-site
interface	I-site
and	O
additional	O
interactions	O
observed	O
with	O
the	O
hexaloop	B-structure_element
,	O
and	O
suggests	O
a	O
preferential	O
binding	O
to	O
the	O
hexaloop	B-structure_element
SL	B-structure_element
RNA	B-chemical
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
.	O

We	O
designed	O
different	O
variants	O
of	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
by	O
point	B-experimental_method
mutagenesis	I-experimental_method
abrogating	O
base	O
pairing	O
in	O
the	O
stem	B-structure_element
region	I-structure_element
,	O
where	O
none	O
,	O
individual	O
,	O
or	O
both	O
SL	B-structure_element
RNA	B-chemical
motifs	O
were	O
mutated	B-experimental_method
to	O
impair	O
Roquin	B-protein
-	I-protein
1	I-protein
binding	O
(	O
Fig	O
.	O
6a	O
).	O

These	O
RNAs	B-chemical
were	O
then	O
tested	O
in	O
EMSAs	B-experimental_method
with	O
the	O
Roquin	B-protein
-	I-protein
1	I-protein
N	O
terminus	O
(	O
residues	O
2	B-residue_range
–	I-residue_range
440	I-residue_range
)	O
(	O
Fig	O
.	O
6b	O
).	O

Gel	B-experimental_method
shift	I-experimental_method
assays	I-experimental_method
show	O
that	O
binding	O
to	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
construct	O
leads	O
to	O
two	O
distinct	O
bands	O
during	O
the	O
titrations	B-experimental_method
,	O
which	O
should	O
reflect	O
binding	O
to	O
one	O
and	O
both	O
RNA	B-chemical
motifs	O
,	O
respectively	O
.	O

Consistent	O
with	O
this	O
,	O
both	O
bands	O
are	O
strongly	O
reduced	O
when	O
mutations	O
are	O
introduced	O
that	O
interfere	O
with	O
the	O
formation	O
of	O
both	O
SLs	B-structure_element
.	O

Notably	O
,	O
among	O
these	O
,	O
the	O
slower	O
migrating	O
band	O
disappears	O
when	O
either	O
of	O
the	O
two	O
SL	B-structure_element
RNA	B-chemical
motifs	O
is	O
altered	O
to	O
impair	O
Roquin	B-protein
binding	O
,	O
indicating	O
an	O
interaction	O
with	O
the	O
remaining	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
SL	B-structure_element
.	O

We	O
thus	O
conclude	O
that	O
Roquin	B-protein
is	O
able	O
to	O
bind	O
to	O
both	O
SL	B-structure_element
RNA	B-chemical
motifs	O
in	O
the	O
context	O
of	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
.	O

Regulation	O
of	O
Ox40	B-protein
expression	O
via	O
two	O
motifs	O
in	O
its	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element

To	O
investigate	O
the	O
role	O
of	O
the	O
new	O
ADE	B-structure_element
-	O
like	O
motif	O
in	O
target	O
mRNA	B-chemical
regulation	O
,	O
we	O
introduced	B-experimental_method
Ox40	B-protein
mRNA	B-chemical
variants	O
harbouring	O
altered	B-protein_state
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
in	O
cells	O
.	O

Considering	O
the	O
close	O
proximity	O
of	O
the	O
ADE	B-structure_element
-	O
like	O
and	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
(	O
Fig	O
.	O
6a	O
),	O
which	O
is	O
essential	O
for	O
Roquin	B-protein
-	O
mediated	O
posttranscriptional	O
regulation	O
of	O
Ox40	B-protein
(	O
ref	O
.)	O
we	O
tested	O
individual	O
contributions	O
and	O
the	O
functional	O
cooperation	O
of	O
the	O
two	O
RNA	B-chemical
elements	O
by	O
deletion	B-experimental_method
and	I-experimental_method
point	I-experimental_method
mutagenesis	I-experimental_method
abrogating	B-protein_state
base	B-bond_interaction
pairing	I-bond_interaction
in	O
the	O
stem	B-structure_element
region	I-structure_element
(	O
Fig	O
.	O
6a	O
,	O
c	O
and	O
Supplementary	O
Fig	O
.	O
8c	O
).	O

Specifically	O
,	O
using	O
retroviruses	B-taxonomy_domain
we	O
introduced	O
Ox40	B-protein
expression	O
constructs	O
placed	O
under	O
the	O
control	O
of	O
different	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
into	O
Roquin	B-protein
-	I-protein
1	I-protein
/	O
2	B-protein
-	O
deficient	O
mouse	B-taxonomy_domain
embryonic	O
fibroblasts	O
.	O

Doxycycline	B-chemical
treatment	O
of	O
cells	O
from	O
this	O
cell	O
line	O
enabled	O
ectopic	O
Roquin	B-protein
-	I-protein
1	I-protein
and	O
co	O
-	O
translational	O
mCherry	O
expression	O
due	O
to	O
the	O
stable	O
integration	O
of	O
an	O
inducible	O
lentiviral	B-taxonomy_domain
vector	O
(	O
Supplementary	O
Fig	O
.	O
8c	O
).	O

The	O
expression	O
of	O
Ox40	B-protein
in	O
cells	O
with	O
and	O
without	O
doxycycline	B-chemical
treatment	O
was	O
then	O
quantified	O
by	O
flow	B-experimental_method
cytometry	I-experimental_method
(	O
Supplementary	O
Fig	O
.	O
8c	O
).	O

Comparing	O
the	O
ratio	O
of	O
Ox40	B-protein
mean	B-evidence
fluorescence	I-evidence
intensities	I-evidence
in	O
cells	O
with	O
and	O
without	O
doxycycline	B-chemical
treatment	O
normalized	O
to	O
the	O
values	O
from	O
cells	O
that	O
expressed	O
Ox40	B-protein
constructs	O
without	B-protein_state
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
revealed	O
a	O
comparable	O
importance	O
of	O
both	O
structural	O
elements	O
(	O
Fig	O
.	O
6c	O
).	O

In	O
fact	O
,	O
only	O
deletion	B-experimental_method
or	I-experimental_method
point	I-experimental_method
mutagenesis	I-experimental_method
of	O
the	O
sequences	O
encoding	O
both	O
structures	O
at	O
the	O
same	O
time	O
(	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
1	B-residue_range
–	I-residue_range
80	I-residue_range
and	O
double	B-protein_state
mut	I-protein_state
)	O
neutralized	O
Roquin	B-protein
-	O
dependent	O
repression	O
of	O
Ox40	B-protein
.	O

In	O
contrast	O
,	O
individual	O
mutations	B-experimental_method
that	O
left	O
the	O
hexaloop	B-structure_element
(	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
1	B-residue_range
–	I-residue_range
120	I-residue_range
or	O
CDE	B-mutant
mut	I-mutant
)	O
or	O
the	O
CDE	B-structure_element
-	O
like	O
triloop	B-structure_element
intact	B-protein_state
still	O
enabled	O
Roquin	B-protein
-	O
dependent	O
repression	O
,	O
which	O
occurred	O
in	O
an	O
attenuated	O
manner	O
compared	O
with	O
the	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
(	O
Fig	O
.	O
6c	O
).	O

To	O
further	O
analyse	O
the	O
functional	O
consequences	O
of	O
Roquin	B-protein
binding	O
to	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
,	O
we	O
also	O
measured	O
mRNA	B-evidence
decay	I-evidence
rates	I-evidence
after	O
introducing	O
the	O
different	O
Ox40	B-protein
constructs	O
into	O
HeLa	O
tet	O
-	O
off	O
cells	O
that	O
allow	O
to	O
turn	O
off	O
transcription	O
from	O
the	O
tetracycline	O
-	O
repressed	O
vectors	O
by	O
addition	O
of	O
doxycycline	B-chemical
(	O
Fig	O
.	O
6d	O
).	O

Quantitative	B-experimental_method
reverse	I-experimental_method
transcriptase	I-experimental_method
–	I-experimental_method
PCR	I-experimental_method
revealed	O
a	O
strong	O
stabilization	O
of	O
the	O
Ox40	B-protein
mRNA	B-chemical
by	O
deletion	B-experimental_method
of	I-experimental_method
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
(	O
CDS	B-structure_element
t1	B-evidence
/	I-evidence
2	I-evidence
=	O
311	O
min	O
vs	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
t1	B-evidence
/	I-evidence
2	I-evidence
=	O
96	O
min	O
).	O

A	O
comparable	O
stabilization	O
was	O
achieved	O
by	O
combined	B-experimental_method
mutation	I-experimental_method
of	O
the	O
CDE	B-structure_element
-	O
like	O
and	O
the	O
ADE	B-structure_element
-	O
like	O
SLs	B-structure_element
(	O
ADE	B-structure_element
/	O
CDE	B-structure_element
-	O
like	O
mut	B-protein_state
t1	B-evidence
/	I-evidence
2	I-evidence
=	O
255	O
min	O
).	O

Individual	O
mutations	B-experimental_method
of	O
either	O
the	O
ADE	B-structure_element
-	O
like	O
or	O
the	O
CDE	B-structure_element
-	O
like	O
SLs	B-structure_element
showed	O
intermediate	O
effects	O
(	O
ADE	B-structure_element
-	O
like	O
mut	B-protein_state
t1	B-evidence
/	I-evidence
2	I-evidence
=	O
170	O
min	O
,	O
CDE	B-structure_element
-	O
like	O
mut	B-protein_state
t1	B-evidence
/	I-evidence
2	I-evidence
=	O
167	O
min	O
),	O
respectively	O
.	O

These	O
findings	O
underscore	O
the	O
importance	O
of	O
both	O
structural	O
motifs	O
and	O
reveal	O
that	O
they	O
have	O
an	O
additive	O
effect	O
on	O
the	O
regulation	O
of	O
Ox40	B-protein
mRNA	B-chemical
expression	O
in	O
cells	O
.	O

Recent	O
structural	B-experimental_method
and	I-experimental_method
functional	I-experimental_method
studies	I-experimental_method
have	O
provided	O
first	O
insight	O
into	O
the	O
RNA	B-chemical
binding	O
of	O
Roquin	B-protein
.	O

Structures	B-evidence
of	O
Roquin	B-protein
bound	B-protein_state
to	I-protein_state
CDE	B-structure_element
SL	B-structure_element
RNAs	B-chemical
indicated	O
mainly	O
shape	O
recognition	O
of	O
the	O
SL	B-structure_element
RNA	B-chemical
in	O
the	O
so	O
-	O
called	O
A	B-site
-	I-site
site	I-site
of	O
the	O
N	B-structure_element
-	I-structure_element
terminal	I-structure_element
region	I-structure_element
of	O
the	O
Roquin	B-protein
protein	O
with	O
no	O
sequence	O
specificity	O
,	O
except	O
the	O
requirement	O
for	O
a	O
pyrimidine	B-structure_element
–	I-structure_element
purine	I-structure_element
–	I-structure_element
pyrimidine	I-structure_element
triloop	I-structure_element
.	O

Considering	O
that	O
the	O
CDE	B-structure_element
RNA	B-chemical
recognition	O
is	O
mostly	O
structure	O
specific	O
and	O
not	O
sequence	O
dependent	O
,	O
a	O
wide	O
spectrum	O
of	O
target	O
mRNA	B-chemical
might	O
be	O
recognized	O
by	O
Roquin	B-protein
.	O

Here	O
we	O
have	O
used	O
SELEX	B-experimental_method
assays	I-experimental_method
to	O
identify	O
a	O
novel	O
RNA	B-structure_element
recognition	I-structure_element
motif	I-structure_element
of	O
Roquin	B-protein
-	I-protein
1	I-protein
,	O
which	O
is	O
present	O
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
and	O
variations	O
of	O
which	O
may	O
be	O
found	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
of	O
many	O
other	O
genes	O
.	O

Our	O
experiments	O
show	O
that	O
this	O
SELEX	B-experimental_method
-	O
derived	O
ADE	B-structure_element
shows	O
functional	O
activity	O
comparable	O
to	O
the	O
previously	O
established	O
CDE	B-structure_element
motif	O
.	O

The	O
ADE	B-structure_element
and	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
adopt	O
SL	B-structure_element
folds	O
with	O
a	O
hexaloop	B-structure_element
instead	O
of	O
a	O
triloop	B-structure_element
.	O

Notably	O
,	O
the	O
recognition	O
of	O
the	O
respective	O
RNA	B-structure_element
-	I-structure_element
helical	I-structure_element
stem	I-structure_element
regions	I-structure_element
by	O
the	O
ROQ	B-structure_element
domain	O
is	O
identical	O
for	O
the	O
triloop	B-structure_element
and	O
hexaloop	B-structure_element
motifs	O
.	O

However	O
,	O
the	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
hexaloops	I-structure_element
in	O
the	O
ADE	B-structure_element
and	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNAs	B-chemical
mediate	O
a	O
number	O
of	O
additional	O
contacts	O
with	O
the	O
helix	B-structure_element
α4	B-structure_element
and	O
strand	B-structure_element
β3	B-structure_element
in	O
the	O
ROQ	B-structure_element
domain	O
that	O
are	O
absent	O
in	O
the	O
triloop	B-structure_element
CDE	B-structure_element
(	O
Fig	O
.	O
3b	O
–	O
f	O
).	O

Of	O
particular	O
importance	O
for	O
the	O
hexaloop	B-structure_element
recognition	O
is	O
Tyr250	B-residue_name_number
,	O
which	O
acts	O
as	O
a	O
stabilizing	O
element	O
for	O
the	O
integrity	O
of	O
a	O
defined	O
loop	B-structure_element
conformation	O
.	O

It	O
stacks	B-bond_interaction
with	O
nucleotides	O
in	O
the	O
hexaloop	B-structure_element
but	O
not	O
the	O
CDE	B-structure_element
triloop	B-structure_element
(	O
Fig	O
.	O
3b	O
,	O
c	O
).	O

The	O
functional	O
role	O
of	O
Tyr250	B-residue_name_number
for	O
ADE	B-structure_element
-	O
mediated	O
mRNA	B-chemical
regulation	O
by	O
Roquin	B-protein
-	I-protein
1	I-protein
is	O
thus	O
explained	O
by	O
our	O
experiments	O
(	O
Fig	O
.	O
5b	O
,	O
c	O
).	O

The	O
preference	O
for	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
hexaloops	I-structure_element
depends	O
on	O
nucleotide	O
-	O
specific	O
interactions	O
of	O
ROQ	B-structure_element
with	O
U10	B-residue_name_number
,	O
U11	B-residue_name_number
and	O
U13	B-residue_name_number
in	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
.	O

Consistent	O
with	O
this	O
,	O
loss	O
of	O
ROQ	B-structure_element
binding	O
is	O
observed	O
on	O
replacement	B-experimental_method
of	O
U11	B-residue_name_number
and	O
U13	B-residue_name_number
by	O
other	O
bases	O
(	O
Table	O
2	O
).	O

In	O
spite	O
of	O
these	O
differences	O
in	O
some	O
aspects	O
of	O
the	O
RNA	B-chemical
recognition	O
,	O
overall	O
features	O
of	O
Roquin	B-protein
targets	O
are	O
conserved	O
in	O
ADE	B-structure_element
and	O
CDE	B-structure_element
-	O
like	O
RNAs	B-chemical
,	O
namely	O
,	O
a	O
crucial	O
role	O
of	O
non	O
-	O
sequence	O
-	O
specific	O
contacts	O
to	O
the	O
RNA	B-chemical
stem	B-structure_element
and	O
mainly	O
shape	O
recognition	O
of	O
the	O
hexa	B-structure_element
-	I-structure_element
and	I-structure_element
triloops	I-structure_element
,	O
respectively	O
.	O

A	O
unique	O
feature	O
of	O
the	O
bound	B-protein_state
RNA	B-chemical
structure	B-evidence
,	O
common	O
to	O
both	O
tri	B-structure_element
-	I-structure_element
and	I-structure_element
hexaloops	I-structure_element
,	O
is	O
the	O
stacking	B-bond_interaction
of	O
a	O
purine	O
base	O
onto	O
the	O
closing	O
base	O
pair	O
(	O
Fig	O
.	O
3b	O
,	O
c	O
).	O

Previous	O
structural	B-evidence
data	I-evidence
and	O
the	O
results	O
presented	O
here	O
therefore	O
suggest	O
that	O
Roquin	B-protein
may	O
recognize	O
additional	O
SL	B-structure_element
RNA	B-chemical
motifs	O
,	O
potentially	O
with	O
larger	O
loops	B-structure_element
.	O

Interestingly	O
,	O
the	O
SELEX	B-experimental_method
-	O
derived	O
motif	O
resembles	O
the	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
motifs	I-structure_element
that	O
were	O
identified	O
recently	O
by	O
Murakawa	O
et	O
al	O
..	O
In	O
their	O
study	O
,	O
several	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
loops	I-structure_element
of	O
various	O
sizes	O
were	O
identified	O
by	O
crosslinking	B-experimental_method
and	I-experimental_method
immunoprecipitation	I-experimental_method
of	O
Roquin	B-protein
-	I-protein
1	I-protein
using	O
PAR	B-experimental_method
-	I-experimental_method
CLIP	I-experimental_method
and	O
the	O
data	O
also	O
included	O
sequences	O
comprising	O
the	O
U	B-structure_element
-	I-structure_element
rich	I-structure_element
hexaloop	I-structure_element
identified	O
in	O
our	O
present	O
work	O
.	O

Most	O
probably	O
,	O
the	O
experimental	O
setup	O
of	O
Murakawa	O
et	O
al	O
.	O
revealed	O
both	O
high	O
-	O
and	O
low	O
-	O
affinity	O
target	O
motifs	O
for	O
Roquin	B-protein
,	O
whereas	O
our	O
structural	B-experimental_method
study	I-experimental_method
reports	O
on	O
a	O
high	O
-	O
affinity	O
binding	O
motif	O
.	O

Notably	O
,	O
Murakawa	O
et	O
al	O
.	O
neither	O
found	O
the	O
Roquin	B-protein
-	O
regulated	O
Ox40	B-protein
nor	O
the	O
Tnf	B-protein
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
,	O
as	O
both	O
genes	O
are	O
not	O
expressed	O
in	O
HEK	O
293	O
cells	O
.	O

However	O
,	O
their	O
newly	O
identified	O
U	O
-	O
rich	O
target	O
SL	B-structure_element
within	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
A20	B-protein
mRNA	B-chemical
supports	O
our	O
conclusion	O
that	O
Roquin	B-protein
can	O
accept	O
alternative	O
target	O
motifs	O
apart	O
from	O
the	O
classical	O
CDE	B-structure_element
triloop	B-structure_element
arrangement	O
.	O

It	O
remains	O
to	O
be	O
seen	O
which	O
exact	O
features	O
govern	O
the	O
recognition	O
of	O
the	O
A20	B-protein
SL	B-structure_element
by	O
Roquin	B-protein
.	O

The	O
regulatory	O
cis	B-structure_element
RNA	I-structure_element
elements	I-structure_element
in	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
may	O
also	O
be	O
targeted	O
by	O
additional	O
trans	O
-	O
acting	O
factors	O
.	O

We	O
have	O
recently	O
identified	O
the	O
endonuclease	B-protein_type
Regnase	B-protein
-	I-protein
1	I-protein
as	O
a	O
cofactor	O
of	O
Roquin	B-protein
function	O
that	O
shares	O
an	O
overlapping	O
set	O
of	O
target	O
mRNAs	B-chemical
.	O

In	O
another	O
study	O
,	O
the	O
overlap	O
in	O
targets	O
was	O
confirmed	O
,	O
but	O
a	O
mutually	O
exclusive	O
regulation	O
was	O
proposed	O
based	O
on	O
studies	O
in	O
lipopolysaccharide	B-chemical
(	O
LPS	B-chemical
)-	O
stimulated	O
myeloid	O
cells	O
.	O

In	O
these	O
cells	O
,	O
Roquin	B-protein
induced	O
mRNA	B-chemical
decay	O
only	O
for	O
translationally	O
inactive	B-protein_state
mRNAs	B-chemical
,	O
while	O
Regnase	B-protein
-	I-protein
1	I-protein
-	O
induced	O
mRNA	B-chemical
decay	O
depended	O
on	O
active	O
translation	O
of	O
the	O
target	O
.	O

In	O
CD4	O
+	O
T	O
cells	O
,	O
Ox40	B-protein
does	O
not	O
show	O
derepression	O
in	O
individual	O
knockouts	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
or	O
Roquin	B-protein
-	I-protein
2	I-protein
encoding	O
genes	O
,	O
but	O
is	O
strongly	O
induced	O
upon	O
combined	O
deficiency	B-experimental_method
of	O
both	O
genes	O
.	O

In	O
addition	O
,	O
conditional	O
deletion	B-experimental_method
of	I-experimental_method
the	O
Regnase	B-protein
-	I-protein
1	I-protein
-	O
encoding	O
gene	O
induced	O
Ox40	B-protein
expression	O
in	O
these	O
cells	O
.	O

Whether	O
induced	O
decay	O
of	O
Ox40	B-protein
mRNA	B-chemical
by	O
Roquin	B-protein
or	O
Regnase	B-protein_type
proteins	O
occurs	O
in	O
a	O
mutually	O
exclusive	O
manner	O
at	O
different	O
points	O
during	O
T	O
-	O
cell	O
activation	O
or	O
shows	O
cooperative	O
regulation	O
will	O
have	O
to	O
await	O
a	O
direct	O
comparison	O
of	O
T	O
cells	O
with	O
single	O
,	O
double	B-experimental_method
and	I-experimental_method
triple	I-experimental_method
knockouts	I-experimental_method
of	O
these	O
genes	O
.	O

However	O
,	O
in	O
cultures	O
of	O
CD4	O
+	O
T	O
cells	O
,	O
Ox40	B-protein
is	O
translated	O
on	O
day	O
4	O
–	O
5	O
and	O
is	O
expressed	O
much	O
higher	O
in	O
T	O
cells	O
with	O
combined	O
deficiency	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
and	O
Roquin	B-protein
-	I-protein
2	I-protein
.	O

At	O
this	O
time	O
point	O
,	O
the	O
short	O
-	O
term	O
inducible	O
reconstitution	B-experimental_method
with	O
WT	B-protein_state
Roquin	B-protein
-	I-protein
1	I-protein
was	O
effective	O
to	O
reduced	O
Ox40	B-protein
expression	O
,	O
demonstrating	O
the	O
regulation	O
of	O
a	O
translationally	O
active	B-protein_state
mRNA	B-chemical
by	O
Roquin	B-protein
-	I-protein
1	I-protein
in	O
T	O
cells	O
(	O
Fig	O
.	O
5c	O
).	O

Recombinant	O
N	O
-	O
terminal	O
protein	O
fragments	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
or	O
Roquin	B-protein
-	I-protein
2	I-protein
bind	O
with	O
comparable	O
affinity	O
to	O
Ox40	B-protein
mRNA	B-chemical
in	O
EMSAs	B-experimental_method
and	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
Ox40	B-protein
is	O
similarly	O
retained	O
by	O
the	O
two	O
recombinant	O
proteins	O
in	O
filter	B-experimental_method
binding	I-experimental_method
assays	I-experimental_method
.	O

Given	O
the	O
almost	O
identical	O
RNA	B-chemical
contacts	O
in	O
both	O
paralogues	O
,	O
we	O
assume	O
a	O
similar	O
recognition	O
of	O
ADE	B-structure_element
and	O
CDE	B-structure_element
motifs	O
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
by	O
both	O
proteins	O
.	O

In	O
contrast	O
,	O
structural	O
details	O
on	O
how	O
Regnase	B-protein
-	I-protein
1	I-protein
can	O
interact	O
with	O
these	O
SL	B-structure_element
RNAs	B-chemical
are	O
currently	O
missing	O
.	O

Surprisingly	O
,	O
transcriptome	O
-	O
wide	O
mapping	O
of	O
Regnase	B-site
-	I-site
1	I-site
-	I-site
binding	I-site
sites	I-site
in	O
crosslinking	B-experimental_method
and	I-experimental_method
immunoprecipitation	I-experimental_method
experiments	I-experimental_method
identified	O
specific	O
triloop	B-structure_element
structures	O
with	O
pyrimidine	B-structure_element
–	I-structure_element
purine	I-structure_element
–	I-structure_element
pyrimidine	I-structure_element
loops	I-structure_element
in	O
3	O
-	O
to	O
7	O
-	O
nt	O
-	O
long	O
stems	B-structure_element
,	O
as	O
well	O
as	O
a	O
novel	O
hexaloop	B-structure_element
structure	O
in	O
the	O
Ptgs2	B-gene
gene	O
.	O

Both	O
were	O
required	O
for	O
Regnase	B-protein
-	I-protein
1	I-protein
-	O
mediated	O
repression	O
.	O

These	O
findings	O
therefore	O
raise	O
the	O
possibility	O
that	O
Regnase	B-protein
-	I-protein
1	I-protein
interacts	O
with	O
ADE	B-structure_element
-	O
like	O
hexaloop	B-structure_element
structures	O
either	O
in	O
a	O
direct	O
or	O
indirect	O
manner	O
.	O

Nevertheless	O
,	O
it	O
becomes	O
clear	O
that	O
composite	O
cis	B-structure_element
-	I-structure_element
elements	I-structure_element
,	O
that	O
is	O
,	O
the	O
presence	O
of	O
several	O
SLs	B-structure_element
as	O
in	O
Ox40	B-protein
or	O
Icos	B-protein
,	O
could	O
attract	O
multiple	O
trans	O
-	O
acting	O
factors	O
that	O
may	O
potentially	O
co	O
-	O
regulate	O
or	O
even	O
act	O
cooperatively	O
to	O
control	O
mRNA	B-chemical
expression	O
through	O
posttranscriptional	O
pathways	O
of	O
gene	O
regulation	O
.	O

The	O
novel	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
loop	B-structure_element
motif	I-structure_element
that	O
we	O
have	O
identified	O
as	O
a	O
bona	O
fide	O
target	O
of	O
Roquin	B-protein
now	O
expands	O
this	O
multilayer	O
mode	O
of	O
co	O
-	O
regulation	O
.	O

We	O
suggest	O
that	O
differential	O
regulation	O
of	O
mRNA	B-chemical
expression	O
is	O
not	O
only	O
achieved	O
through	O
multiple	O
regulators	O
with	O
individual	O
preferences	O
for	O
a	O
given	O
motif	O
or	O
variants	O
thereof	O
,	O
but	O
that	O
regulators	O
may	O
also	O
identify	O
and	O
use	O
distinct	O
motifs	O
,	O
as	O
long	O
as	O
they	O
exhibit	O
some	O
basic	O
features	O
regarding	O
shape	O
,	O
size	O
and	O
sequence	O
.	O

The	O
presence	O
of	O
distinct	O
motifs	O
in	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
offers	O
a	O
broader	O
variability	O
for	O
gene	O
regulation	O
by	O
RNA	B-chemical
cis	B-structure_element
elements	I-structure_element
.	O

Their	O
accessibility	O
can	O
be	O
modulated	O
by	O
trans	O
-	O
acting	O
factors	O
that	O
may	O
bind	O
regulatory	O
motifs	O
,	O
unfold	O
higher	O
-	O
order	O
structures	O
in	O
the	O
RNA	B-chemical
or	O
maintain	O
a	O
preference	O
for	O
duplex	O
structures	O
as	O
was	O
shown	O
recently	O
for	O
mRNAs	B-chemical
that	O
are	O
recognized	O
by	O
Staufen	B-protein
-	I-protein
1	I-protein
(	O
ref	O
.).	O

In	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
the	O
Ox40	B-protein
mRNA	B-chemical
,	O
we	O
find	O
one	O
ADE	B-structure_element
-	O
like	O
and	O
one	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
,	O
with	O
similar	O
binding	O
to	O
the	O
ROQ	B-structure_element
domain	O
.	O

The	O
exact	O
stoichiometry	O
of	O
Roquin	B-protein
bound	B-protein_state
to	I-protein_state
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
is	O
unknown	O
.	O

The	O
recently	O
identified	O
secondary	B-site
binding	I-site
site	I-site
for	O
dsRNA	B-chemical
in	O
Roquin	B-protein
(	O
B	B-site
-	I-site
site	I-site
)	O
could	O
potentially	O
allow	O
for	O
simultaneous	O
binding	O
of	O
dsRNA	B-chemical
and	O
thereby	O
promote	O
engagement	O
of	O
Roquin	B-protein
and	O
target	O
RNAs	B-chemical
before	O
recognition	O
of	O
high	O
-	O
affinity	B-evidence
SLs	B-structure_element
.	O

In	O
this	O
respect	O
,	O
it	O
is	O
interesting	O
to	O
note	O
that	O
symmetry	O
-	O
related	O
RNA	B-chemical
molecules	O
of	O
both	O
Tnf	B-protein
CDE	B-structure_element
and	O
ADE	B-structure_element
SL	B-structure_element
RNAs	B-chemical
are	O
found	O
in	O
the	O
respective	O
crystal	B-evidence
lattice	I-evidence
in	O
a	O
position	O
that	O
corresponds	O
to	O
the	O
recognition	O
of	O
dsRNA	B-chemical
in	O
the	O
B	B-site
site	I-site
.	O

This	O
opens	O
the	O
possibility	O
that	O
one	O
Roquin	B-protein
molecule	O
may	O
cluster	O
two	O
motifs	O
in	O
a	O
given	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
and	O
/	O
or	O
cluster	O
motifs	O
from	O
distinct	O
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
to	O
enhance	O
downstream	O
processing	O
.	O

Interestingly	O
,	O
two	O
SL	B-structure_element
RNA	B-chemical
elements	O
that	O
resemble	O
bona	O
fide	O
ligands	O
of	O
Roquin	B-protein
have	O
also	O
been	O
identified	O
in	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
of	O
the	O
Nfkbid	B-protein
mRNA	B-chemical
.	O

We	O
therefore	O
hypothesize	O
that	O
the	O
combination	O
of	O
multiple	O
binding	B-site
sites	I-site
may	O
be	O
more	O
commonly	O
used	O
to	O
enhance	O
the	O
functional	O
activity	O
of	O
Roquin	B-protein
.	O

At	O
the	O
same	O
time	O
,	O
the	O
combination	O
of	O
cis	B-structure_element
elements	I-structure_element
may	O
be	O
important	O
for	O
differential	O
gene	O
regulation	O
,	O
as	O
composite	O
cis	B-structure_element
elements	I-structure_element
with	O
lower	O
affinity	B-evidence
may	O
be	O
less	O
sensitive	O
to	O
Roquin	B-protein
.	O

This	O
will	O
lead	O
to	O
less	O
effective	O
repression	O
in	O
T	O
cells	O
when	O
antigen	O
recognition	O
is	O
of	O
moderate	O
signal	O
strength	O
and	O
only	O
incomplete	O
cleavage	O
of	O
Roquin	B-protein
by	O
MALT1	B-protein
occurs	O
.	O

For	O
understanding	O
the	O
intricate	O
complexity	O
of	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
regulation	O
,	O
future	O
work	O
will	O
be	O
necessary	O
by	O
combining	O
large	O
-	O
scale	O
approaches	O
,	O
such	O
as	O
cross	B-experimental_method
-	I-experimental_method
linking	I-experimental_method
and	I-experimental_method
immunoprecipitation	I-experimental_method
experiments	I-experimental_method
to	O
identify	O
RNA	B-site
-	I-site
binding	I-site
sites	I-site
,	O
and	O
structural	B-experimental_method
biology	I-experimental_method
to	O
dissect	O
the	O
underlying	O
molecular	O
mechanisms	O
.	O

SELEX	B-experimental_method
identifies	O
a	O
novel	O
SL	B-structure_element
RNA	B-chemical
ligand	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
.	O

(	O
a	O
)	O
Enriched	O
hexamers	O
that	O
were	O
found	O
by	O
Roquin	B-protein
-	I-protein
1	I-protein
N	O
terminus	O
(	O
residues	O
2	B-residue_range
–	I-residue_range
440	I-residue_range
)	O
or	O
Roquin	B-mutant
-	I-mutant
1	I-mutant
M199R	I-mutant
N	O
terminus	O
(	O
residues	O
2	B-residue_range
–	I-residue_range
440	I-residue_range
)	O
(	O
see	O
also	O
Supplementary	O
Fig	O
.	O
1	O
).	O
(	O
b	O
)	O
An	O
ADE	B-structure_element
sequence	O
motif	O
in	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
closely	O
resembles	O
the	O
MEME	B-experimental_method
motif	O
found	O
in	O
SELEX	B-experimental_method
-	O
enriched	O
RNA	B-chemical
sequences	O
.	O

(	O
c	O
)	O
Conservation	O
of	O
the	O
motif	O
found	O
in	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTRs	I-structure_element
for	O
various	O
species	O
as	O
indicated	O
.	O

rn5	B-gene
is	O
the	O
fifth	O
assembly	O
version	O
of	O
the	O
rat	B-taxonomy_domain
(	O
Rattus	B-species
novegicus	I-species
).	O
(	O
d	O
)	O
Schematic	O
representation	O
of	O
the	O
predicted	O
SELEX	B-experimental_method
-	O
derived	O
consensus	O
SL	B-structure_element
,	O
ADE	B-structure_element
and	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
hexaloop	B-structure_element
SL	B-structure_element
.	O

The	O
broken	O
line	O
between	O
the	O
G	O
–	O
G	O
base	O
pair	O
in	O
the	O
ADE	B-structure_element
SL	B-structure_element
indicates	O
a	O
putative	O
non	B-bond_interaction
-	I-bond_interaction
Watson	I-bond_interaction
–	I-bond_interaction
Crick	I-bond_interaction
pairing	I-bond_interaction
.	O

The	O
Ox40	B-protein
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
and	O
the	O
Tnf	B-protein
CDE	B-structure_element
SL	B-structure_element
are	O
shown	O
for	O
comparison	O
.	O

NMR	B-experimental_method
analysis	O
of	O
the	O
SL	B-structure_element
RNAs	B-chemical
used	O
in	O
this	O
study	O
.	O

Imino	O
proton	O
regions	O
of	O
one	O
-	O
dimensional	O
1H	B-experimental_method
NMR	I-experimental_method
spectra	B-evidence
of	O
(	O
a	O
)	O
the	O
ADE	B-structure_element
SL	B-structure_element
(	O
b	O
),	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
and	O
(	O
c	O
)	O
the	O
Ox40	B-protein
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
are	O
shown	O
for	O
free	B-protein_state
RNAs	B-chemical
(	O
black	O
)	O
and	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
Roquin	B-protein
-	I-protein
1	I-protein
ROQ	B-structure_element
domain	O
(	O
red	O
).	O

The	O
respective	O
SL	B-structure_element
RNAs	B-chemical
and	O
their	O
base	O
pairs	O
are	O
indicated	O
.	O

Red	O
asterisks	O
indicate	O
NMR	B-experimental_method
signals	O
of	O
the	O
protein	O
.	O

Green	O
lines	O
in	O
the	O
secondary	O
structure	O
schemes	O
on	O
the	O
left	O
refer	O
to	O
visible	O
imino	O
NMR	B-experimental_method
signals	B-evidence
and	O
thus	O
experimental	O
confirmation	O
of	O
the	O
base	O
pairs	O
indicated	O
.	O

The	O
dotted	O
green	O
line	O
between	O
G6	B-residue_name_number
and	O
G15	B-residue_name_number
in	O
a	O
highlights	O
a	O
G	B-residue_name
–	O
G	B-residue_name
base	O
pair	O
.	O

Structure	B-evidence
of	O
the	O
Roquin	B-protein
-	I-protein
1	I-protein
ROQ	B-structure_element
domain	O
bound	B-protein_state
to	I-protein_state
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
RNA	B-chemical
.	O

(	O
a	O
)	O
Cartoon	O
presentation	O
of	O
the	O
crystal	B-evidence
structure	I-evidence
of	O
the	O
ROQ	B-structure_element
domain	O
(	O
residues	O
174	B-residue_range
–	I-residue_range
325	I-residue_range
;	O
blue	O
)	O
and	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
(	O
magenta	O
).	O

Selected	O
RNA	B-chemical
bases	O
and	O
protein	O
secondary	O
structure	O
elements	O
are	O
labelled	O
.	O

(	O
b	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
bases	O
in	O
the	O
RNA	B-chemical
hexaloop	B-structure_element
are	O
shown	O
in	O
magenta	O
)	O
and	O
(	O
c	O
)	O
the	O
previously	O
reported	O
structure	B-evidence
of	O
the	O
ROQ	B-complex_assembly
-	I-complex_assembly
Tnf	I-complex_assembly
CDE	I-complex_assembly
complex	O
(	O
bases	O
of	O
the	O
triloop	O
RNA	B-chemical
are	O
shown	O
in	O
green	O
).	O

Only	O
RNA	B-site
-	I-site
interacting	I-site
residues	I-site
that	O
are	O
different	O
in	O
both	O
structures	B-evidence
are	O
shown	O
.	O

Both	O
protein	O
chains	O
and	O
remaining	O
parts	O
of	O
both	O
RNAs	B-chemical
are	O
shown	O
in	O
grey	O
and	O
protein	O
residue	O
side	O
chains	O
are	O
shown	O
in	O
turquoise	O
.	O
(	O
d	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
contacts	O
between	O
the	O
ROQ	B-structure_element
domain	O
and	O
nucleotides	O
U11	B-residue_name_number
and	O
U13	B-residue_name_number
of	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
RNA	B-chemical
.	O

The	O
nucleotides	O
interact	O
with	O
the	O
C	O
-	O
terminal	O
end	O
of	O
helix	B-structure_element
α4	B-structure_element
(	O
Tyr250	B-residue_name_number
and	O
Ser253	B-residue_name_number
)	O
and	O
the	O
N	O
-	O
terminal	O
part	O
of	O
strand	B-structure_element
β3	B-structure_element
(	O
Phe255	B-residue_name_number
and	O
Val257	B-residue_name_number
).	O

The	O
protein	O
chain	O
is	O
shown	O
in	O
turquoise	O
and	O
the	O
RNA	B-chemical
is	O
shown	O
in	O
grey	O
.	O

(	O
e	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
contacts	O
between	O
the	O
ROQ	B-structure_element
domain	O
and	O
nucleotides	O
U10	B-residue_name_number
,	O
U11	B-residue_name_number
and	O
U13	B-residue_name_number
in	O
the	O
RNA	B-chemical
hexaloop	B-structure_element
.	O

U11	B-residue_name_number
and	O
U13	B-residue_name_number
contact	O
the	O
C	O
-	O
terminal	O
end	O
of	O
helix	B-structure_element
α4	B-structure_element
:	O
residues	O
Tyr250	B-residue_name_number
and	O
Gln247	B-residue_name_number
.	O

The	O
side	O
chain	O
of	O
Tyr250	B-residue_name_number
makes	O
hydrophobic	B-bond_interaction
interactions	I-bond_interaction
with	O
the	O
pyrimidine	O
side	O
chain	O
of	O
U10	B-residue_name_number
on	O
one	O
side	O
and	O
U11	B-residue_name_number
on	O
the	O
other	O
side	O
.	O

Lys259	B-residue_name_number
interacts	O
with	O
the	O
phosphate	O
groups	O
of	O
U10	B-residue_name_number
and	O
U11	B-residue_name_number
.	O

(	O
f	O
)	O
Close	O
-	O
up	O
view	O
of	O
the	O
hydrophobic	B-bond_interaction
interaction	I-bond_interaction
between	O
Val257	B-residue_name_number
and	O
U11	B-residue_name_number
,	O
as	O
well	O
as	O
the	O
double	O
hydrogen	B-bond_interaction
bond	I-bond_interaction
of	O
Lys259	B-residue_name_number
with	O
phosphate	O
groups	O
of	O
U10	B-residue_name_number
and	O
U11	B-residue_name_number
.	O

NMR	B-experimental_method
analysis	O
of	O
ROQ	B-structure_element
domain	O
interactions	O
with	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
hexaloop	B-structure_element
RNA	B-chemical
.	O

(	O
a	O
)	O
Overlay	B-experimental_method
of	O
1H	B-experimental_method
,	I-experimental_method
15N	I-experimental_method
HSQC	I-experimental_method
spectra	B-evidence
of	O
either	O
the	O
free	B-protein_state
ROQ	B-structure_element
domain	O
(	O
171	B-residue_range
–	I-residue_range
326	I-residue_range
,	O
black	O
)	O
or	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
stoichiometric	O
amounts	O
of	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
red	O
).	O

(	O
b	O
)	O
Plot	O
of	O
chemical	B-evidence
shift	I-evidence
change	I-evidence
versus	O
residue	O
number	O
in	O
the	O
ROQ	B-structure_element
domain	O
(	O
residues	O
171	B-residue_range
–	I-residue_range
326	I-residue_range
)	O
from	O
a	O
.	O
Grey	O
negative	O
bars	O
indicate	O
missing	O
assignments	O
in	O
one	O
of	O
the	O
spectra	B-evidence
.	O

Gaps	O
indicate	O
prolines	B-residue_name
.	O

(	O
c	O
)	O
Overlay	B-experimental_method
of	O
the	O
ROQ	B-structure_element
domain	O
alone	B-protein_state
(	O
black	O
)	O
or	O
in	B-protein_state
complex	I-protein_state
with	I-protein_state
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
red	O
)	O
or	O
the	O
Ox40	B-protein
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
green	O
).	O

Mutational	B-experimental_method
analysis	I-experimental_method
of	O
Roquin	B-protein
-	I-protein
1	I-protein
-	O
interactions	O
with	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
and	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
.	O

(	O
a	O
)	O
EMSA	B-experimental_method
assay	I-experimental_method
comparing	O
binding	O
of	O
the	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
and	O
of	O
the	O
Y250A	B-mutant
mutant	B-protein_state
ROQ	B-structure_element
domain	O
for	O
binding	O
to	O
the	O
Ox40	B-protein
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
(	O
left	O
)	O
or	O
the	O
previously	O
described	O
Tnf	B-protein
CDE	B-structure_element
SL	B-structure_element
(	O
right	O
).	O

A	O
comparison	O
of	O
further	O
mutants	O
is	O
shown	O
in	O
Supplementary	O
Fig	O
.	O
4	O
.	O
(	O
b	O
)	O
Schematic	O
overview	O
of	O
the	O
timeline	O
used	O
for	O
the	O
reconstitution	O
experiment	O
shown	O
in	O
c	O
.	O
(	O
c	O
)	O
Flow	B-experimental_method
cytometry	I-experimental_method
of	O
Ox40	B-protein
and	O
Icos	B-protein
surface	O
expression	O
on	O
CD4	O
+	O
Th1	O
cells	O
from	O
Rc3h1	B-gene
/	O
2fl	B-gene
/	O
fl	B-gene
;	O
Cd4	O
-	O
Cre	O
-	O
ERT2	O
;	O
rtTA	O
mice	B-taxonomy_domain
treated	O
with	O
tamoxifen	B-chemical
(+	O
tam	O
)	O
to	O
induce	O
Rc3h1	B-gene
/	O
2fl	B-gene
/	O
fl	B-gene
deletion	B-experimental_method
or	O
left	O
untreated	O
(−	O
tam	O
).	O

The	O
cells	O
were	O
then	O
either	O
left	O
untransduced	O
(	O
UT	O
)	O
or	O
were	O
transduced	O
with	O
retrovirus	B-taxonomy_domain
containing	O
a	O
doxycycline	B-chemical
-	O
inducible	O
cassette	O
,	O
to	O
express	O
Roquin	B-protein
-	I-protein
1	I-protein
WT	B-protein_state
,	O
Roquin	B-protein
-	I-protein
1	I-protein
Y250A	B-mutant
or	O
Roquin	B-protein
-	I-protein
1	I-protein
K220A	B-mutant
,	O
K239A	B-mutant
and	O
R260A	B-mutant
mutants	B-protein_state
(	O
see	O
also	O
Supplementary	O
Fig	O
.	O
5	O
).	O

Functional	O
importance	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
target	O
motifs	O
in	O
cells	O
.	O

(	O
a	O
)	O
Overview	O
of	O
the	O
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
and	O
truncated	B-protein_state
/	O
mutated	B-protein_state
versions	O
thereof	O
as	O
used	O
for	O
EMSA	B-experimental_method
assays	O
in	O
b	O
and	O
the	O
expression	O
experiments	O
of	O
Ox40	B-protein
in	O
c	O
and	O
d	O
.	O
(	O
b	O
)	O
EMSA	B-experimental_method
experiments	O
probing	O
the	O
interaction	O
between	O
the	O
Roquin	B-protein
-	I-protein
1	I-protein
N	O
-	O
terminal	O
region	O
(	O
residues	O
2	B-residue_range
–	I-residue_range
440	I-residue_range
)	O
and	O
either	O
the	O
complete	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
Ox40	B-protein
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
or	O
versions	O
with	O
mutations	B-experimental_method
of	O
the	O
CDE	B-structure_element
-	O
like	O
SL	B-structure_element
,	O
the	O
ADE	B-structure_element
-	O
like	O
SL	B-structure_element
or	O
both	O
SLs	B-structure_element
(	O
see	O
a	O
).	O

It	O
is	O
noteworthy	O
that	O
the	O
higher	O
bands	O
observed	O
at	O
large	O
protein	O
concentrations	O
are	O
probably	O
additional	O
nonspecific	O
,	O
lower	O
-	O
affinity	O
interactions	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
with	O
the	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
or	O
protein	O
aggregates	O
.	O

(	O
c	O
)	O
Relative	O
Ox40	B-protein
MFI	B-evidence
normalized	I-evidence
to	I-evidence
expression	I-evidence
levels	I-evidence
from	O
the	O
Ox40	B-protein
CDS	B-structure_element
construct	O
.	O

Error	O
bars	O
show	O
s	O
.	O
d	O
.	O
of	O
seven	O
(	O
CDS	B-structure_element
,	O
1	B-residue_range
–	I-residue_range
40	I-residue_range
,	O
1	B-residue_range
–	I-residue_range
80	I-residue_range
,	O
1	B-residue_range
–	I-residue_range
120	I-residue_range
and	O
full	B-protein_state
-	I-protein_state
length	I-protein_state
),	O
six	O
(	O
ADE	B-structure_element
-	O
like	O
mut	B-protein_state
and	O
CDE	B-structure_element
mut	B-protein_state
)	O
or	O
three	O
(	O
double	B-protein_state
mut	I-protein_state
)	O
independent	O
experiments	O
.	O

Statistical	O
significance	O
was	O
calculated	O
by	O
one	B-experimental_method
-	I-experimental_method
way	I-experimental_method
analysis	I-experimental_method
of	I-experimental_method
variance	I-experimental_method
(	O
ANOVA	B-experimental_method
)	O
Kruskal	B-experimental_method
–	I-experimental_method
Wallis	I-experimental_method
test	I-experimental_method
followed	O
by	O
Dunn	B-experimental_method
’	I-experimental_method
s	I-experimental_method
multiple	I-experimental_method
comparison	I-experimental_method
test	I-experimental_method
(**	O
P	O
<	O
0	O
.	O
01	O
).	O

(	O
d	O
)	O
mRNA	B-evidence
decay	I-evidence
curves	I-evidence
of	O
Hela	O
Tet	O
-	O
Off	O
cells	O
stably	O
transduced	O
with	O
retroviruses	B-taxonomy_domain
expressing	O
Ox40	B-protein
CDS	B-structure_element
without	O
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
(	O
CDS	B-structure_element
,	O
red	O
line	O
),	O
Ox40	B-protein
CDS	B-structure_element
with	O
its	O
wild	B-protein_state
-	I-protein_state
type	I-protein_state
3	B-structure_element
′-	I-structure_element
UTR	I-structure_element
(	O
full	B-protein_state
length	I-protein_state
,	O
black	O
line	O
),	O
Ox40	B-protein
full	B-protein_state
length	I-protein_state
with	O
mutated	B-protein_state
ADE	B-structure_element
-	O
like	O
motif	O
(	O
ADE	B-structure_element
-	O
like	O
mut	B-protein_state
,	O
grey	O
line	O
),	O
Ox40	B-protein
full	B-protein_state
length	I-protein_state
with	O
mutated	B-protein_state
CDE	B-structure_element
-	O
like	O
motif	O
(	O
CDE	B-structure_element
-	O
like	O
mut	B-protein_state
,	O
green	O
line	O
)	O
or	O
Ox40	B-protein
full	B-protein_state
length	I-protein_state
with	O
mutated	B-protein_state
ADE	B-structure_element
and	O
CDE	B-structure_element
motifs	O
(	O
Double	B-protein_state
mut	I-protein_state
,	O
blue	O
line	O
).	O

mRNA	B-evidence
half	I-evidence
-	I-evidence
life	I-evidence
times	I-evidence
were	O
calculated	O
with	O
Graph	O
Pad	O
Prism	O
.	O

Data	B-evidence
collection	I-evidence
and	I-evidence
refinement	I-evidence
statistics	I-evidence
.	O

ROQ	B-structure_element
-	O
Ox40ADE	B-protein
-	O
like	O
SL	B-structure_element
ROQ	B-structure_element
-	O
ADE	B-structure_element
SL	B-structure_element
Data	O
collection	O
space	O
group	O
P21212	O
P212121	O
Cell	O
dimensions	O
a	O
,	O
b	O
,	O
c	O
(	O
Å	O
)	O
89	O
.	O
66	O
,	O
115	O
.	O
79	O
,	O
42	O
.	O
61	O
72	O
.	O
90	O
,	O
89	O
.	O
30	O
,	O
144	O
.	O
70	O
α	O
,	O
β	O
,	O
γ	O
(°)	O
90	O
,	O
90	O
,	O
90	O
90	O
,	O
90	O
,	O
90	O
Resolution	O
(	O
Å	O
)	O
50	O
–	O
2	O
.	O
23	O
(	O
2	O
.	O
29	O
–	O
2	O
.	O
23	O
)	O
50	O
–	O
3	O
.	O
0	O
(	O
3	O
.	O
08	O
–	O
3	O
.	O
00	O
)	O
Rmerge	O
5	O
.	O
9	O
(	O
68	O
.	O
3	O
)	O
14	O
.	O
8	O
(	O
93	O
.	O
8	O
)	O
I	O
/	O
σI	O
14	O
.	O
9	O
(	O
2	O
.	O
1	O
)	O
16	O
.	O
7	O
(	O
3	O
.	O
1	O
)	O
Completeness	O
(%)	O
98	O
.	O
7	O
(	O
97	O
.	O
7	O
)	O
99	O
.	O
9	O
(	O
99	O
.	O
9	O
)	O
Redundancy	O
3	O
.	O
9	O
(	O
3	O
.	O
7	O
)	O
13	O
.	O
2	O
(	O
12	O
.	O
7	O
)	O
Refinement	O
Resolution	O
(	O
Å	O
)	O
2	O
.	O
23	O
3	O
.	O
00	O
No	O
.	O
reflections	O
21	O
,	O
018	O
18	O
,	O
598	O
Rwork	O
/	O
Rfree	O
21	O
.	O
8	O
/	O
25	O
.	O
7	O
18	O
.	O
6	O
/	O
23	O
.	O
4	O
No	O
.	O
atoms	O
Protein	O
2	O
,	O
404	O
4	O
,	O
820	O
Ligand	O
/	O
ion	O
894	O
1	O
,	O
708	O
Water	O
99	O
49	O
B	O
-	O
factor	O
overall	O
47	O
.	O
2	O
60	O
.	O
4	O
Root	B-evidence
mean	I-evidence
squared	I-evidence
deviations	I-evidence
Bond	O
lengths	O
(	O
Å	O
)	O
0	O
.	O
006	O
0	O
.	O
014	O
Bond	O
angles	O
(°)	O
1	O
.	O
07	O
1	O
.	O
77	O
Ramachandran	O
plot	O
Most	O
favoured	O
(%)	O
98	O
.	O
6	O
99	O
.	O
8	O
Additional	O
allowed	O
(%)	O
1	O
.	O
4	O
0	O
.	O
2	O

ADE	B-structure_element
,	O
alternative	B-structure_element
decay	I-structure_element
element	I-structure_element
;	O
CDE	B-structure_element
,	O
constitutive	B-structure_element
decay	I-structure_element
element	I-structure_element
;	O
SL	B-structure_element
,	O
stem	B-structure_element
loop	I-structure_element
.	O

For	O
each	O
data	O
set	O
,	O
only	O
one	O
crystal	B-evidence
has	O
been	O
used	O
.	O

KD	B-evidence
for	O
selected	O
RNAs	B-chemical
obtained	O
from	O
SPR	B-experimental_method
measurements	I-experimental_method
with	O
immobilized	O
ROQ	B-structure_element
domain	O
of	O
Roquin	B-protein
-	I-protein
1	I-protein
.	O