Chondrolectin is a direct target of miR-431 and is decreased in SMN deficient motor neurons. (
A
) qRT-PCR of Chodl confirms reduced expression in SMN knockdown motor neurons.
N
= 8 from 4 experiments, ***
P
< 0.001, unpaired two-tailed Student’s
t
-test. (
B
) qRT-PCR in cortical neurons shows Chodl is not reduced.
N
= 7 from 3 experiments. (
C
) Schematic depiction of the evolutionary conservation of the miR-431 binding site (highlighted in red) in the
Chodl
3′UTR in mammals. The site is predicted by both the TargetScan −0.29 and PITA −10.17 algorithms. (
D
) Transfection of anti-miR-431 in motor neurons increases
Chodl
expression.
N
= 6 from 3 experiments, **
P
< 0.01, ****
P
< 0.0001, unpaired two-tailed Student’s
t
-test. (
E
) Transfection of a miR-431 mimic in motor neurons decreases expression of
Chodl
.
N
= 8 from 4 experiments. *
P
< 0.05, unpaired two-tailed Student’s
t
-test. (
F
) Schematic of control and mutated (highlighted in red) luciferase constructs of the binding site in the
Chodl
3′UTR. (
G
) Relative
Renilla
/firefly luciferase expression following miR-431 mimic expression in Chodl3′UTR and Chodl3′UTRmut psicheck-2 transfected HEK293 cells at 24 h. Wild-type Chodl3′UTR
N
= 7 from 3 experiments. Chodl3′UTRmut
N
= 5 from two experiments. Significance was determined by two way ANOVA ***
P
< 0.001. Data are represented as mean ± SEM.