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to further explore the mode of action of streptomycin-mediated
inhibition. Addition of streptomycin led to thermal stabilization of
pre-miR-21, indicating an interaction with the miRNA precursor
that may interfere with miRNA maturation (Fig.
6
). When screened
by RT-PCR against an array of nine miRNAs the streptomycin was
found to be relatively specifi c, with only miR-27a also being
downregulated.
Further docking studies between aminoglycosides and pre-
miR-21 revealed direct hydrogen bonding between streptose
groups and the inner walls of the RNA-binding pocket close to the
stem-loop. Given this interaction, researchers chose to investigate
the cleavage activity of Dicer after the addition of streptomycin.
The presence of streptomycin did selectively decrease the levels of
mature miR-21, however only when a specifi c bulge near the ter-
minal loop was available for streptomycin binding. To probe the
effects of streptomycin binding in vivo, a fi refl y luciferase reporter
with the 3
UTR of PDC4 wild-type pre-miR-21 and mutant pre-
miRNA-21 with a deleted bulge were employed. Upon addition of
streptomycin, a 1.2-fold increase in luciferase was observed when
comparing the wild-type and mutant pre-miRNAs revealing the
necessity of the bulge for binding and Dicer processing. Since
PDCD4 plays a role in inducing apoptosis, researchers investigated
the effects of streptomycin-mediated inhibition of miRNA on cell
viability. Streptomycin-treated MCF-7 cancer cells known to over-
express miR-21 showed an increase in apoptosis consistent with an
increase in PDCD4 levels, while Jurkat cells possessing normal
miR-21 levels were unaffected (Fig.
6
). Ultimately, streptomycin
effi ciently represses mature miR-21 levels through direct binding
to precursor molecules, thus interfering with Dicer cleavage.
′
Fig. 6
Streptomycin-modulated inhibition of pre-miRNA maturation. (
a
) Principle of the assay, as streptomycin
binding to pre-miRNA inhibits Dicer processing to the mature miRNA, preventing gene silencing. (
b
) FACS
analysis of two cell lines treated with streptomycin or dihydrostreptomycin. MCF-7 cells with misregulated
miRNA levels exhibit a higher degree of apoptosis when treated with streptomycin, indicating its effect on the
miRNA maturation pathway. Healthy JURKAT cells are not affected by streptomycin treatment and thus exhibit
normal levels of apoptosis. Image adapted with permission from Bose D et al. (2012) Angew Chem Int Ed Engl
