Biology Reference
In-Depth Information
Though streptomycin appears to be specifi c for miR-21, other
small molecules may be found in a larger screen. This exciting
example demonstrates that small-molecule binding of pre-miRNAs
is indeed biologically relevant and has the potential to inhibit Dicer
processing and further miRNA maturation, ultimately modulating
the function of the miRNA within the cell. Future studies must be
conducted to further optimize the specifi city and ascertain if the
streptomycin is engaging in off-target interactions within the com-
plex cellular networks.
Another method for inhibiting miRNA maturation through
precursor targeting involves the use of morpholino oligonucle-
otides [
52
]. These antisense molecules provide a quick method to
knock down miRNA during embryonic development and can be
easily employed in model organisms, e.g., zebra fi sh. However,
these antisense agents must also be used with some degree of
caution as off-target phenotypes can often be observed. Injection
of morpholinos complementary to mature miRNAs suppresses
miRNA levels for up to 4 days. Using GFP reporters fused to
specifi c miRNA target sites, Plasterk and co-workers found that
addition of miRNA silenced the GFP signal, as expected. However,
addition of a miRNA duplex and the complementary morpholino
restores the GFP reporter, revealing that morpholinos can block
the activity of mature miRNA duplexes. Since the addition of
morpholino oligonucleotides did not alter miRNA isolation or
stability, the interaction appears to inhibit miRNA maturation.
As previously noted, the use of antisense oligonucleotides
often results in off-target effects resulting in a different phenotype
than the desired knockout. To compare the effects of morpholinos,
several morpholinos were designed to target the Drosha and Dicer
cleavage sites of precursor miRNAs. These precursor-targeting
morpholinos were extremely effective at inducing a reduction in
the cellular levels of certain miRNAs, such as miR-205. However
some miRNAs, such as miR-30c, were only inhibited by morpho-
linos targeting mature miR-30c and not the morpholinos against
miR-30c precursors. The discrepancy may occur as some miRNAs
display more sequence variability and may not be equally suscepti-
ble to morpholinos designed to target precursor molecules (Fig.
7
).
Utilizing a GFP construct with pri-miR-205 in the 3
UTR,
researchers found that the addition of morpholinos resulted in an
accumulation of primary miRNA consistent with the inhibition of
Drosha activity.
Further work with morpholinos centered on the inhibition of
miR-375, a miRNA that has two copies in the zebra fi sh genome
and is expressed in the pancreatic islet as well as the pituitary gland.
Multiple morpholinos were designed to target pri-miR-375-1, pri-
miR-375-2, or mature miR-375. The morpholinos targeting miRNA
precursors were able to interfere with Drosha or Dicer cleavage
steps, thus decreasing the levels of mature miR-375. In comparison,
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