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revealed that the morpholino-injected embryos displayed scattered
islet cells resulting in drastic changes in insulin expression. Close
monitoring of morpholino-injected embryos showed that loss of
miR-375 leads to malformation of the endocrine pancreas in zebra
fi sh. Thus, morpholinos can selectively target mature miRNA
sequences or be used to inhibit Drosha or Dicer cleavage through
targeting of precursor molecules. This effi cient technique allows
for reliable knockdown of various miRNAs during the early stages
of zebra fi sh development. This study demonstrates the impor-
tance of miRNA maturation in vivo and that antisense agents are
capable of targeting not only the function of mature miRNA but
also the processing by both Dicer and Drosha enzymes.
Based on the convergence of the miRNA biogenesis pathway on
the same set of processing enzymes irrespective of the RNA, non-
specifi c targeting of miRNA maturation and function is relatively
easier to achieve than the previously discussed specifi c targeting.
Consequently, there are many more examples of both small-
molecule and biomacromolecular modulators of the processing of
pre-miRNA. This is especially true in vitro where direct assays have
been developed with the isolated components of the maturation
pathway to ensure modulation of their function by the effector
molecule.
3.3 Inhibition of
Pre-miRNA In Vitro
Without Assessing
Specifi city
One such in vitro assay has been developed in the Arenz laboratory
to monitor the ability of Dicer to process pre-miRNA into its active
mature miRNA analog [
53
,
54
]. The fl uorescence-based assay was
developed using a pre-let-7 probe due to its prevalence in miRNA
studies and its well-defi ned Dicer-mediated cleavage. This probe
was modifi ed to contain a 5
3.3.1 Inhibition of
Pre-miRNA Maturation
by Peptides
dabcyl
quencher (Fig.
8
), affording fl uorescence quenching when the
properly folded hairpin pre-miRNA is formed; however, upon
Dicer maturation of the pre-miRNA the proximity of the fl uoro-
phore and the quencher is lost, resulting in a fl uorescence signal.
Incubation of the probe with either recombinant Dicer or cellular
lysate containing Dicer in vitro resulted in a three- to tenfold
increase in fl uorescence. With the assay fi rmly established, inhibi-
tors could be screened for a decrease in fl uorescence signal corre-
sponding to the prevention of pre-miRNA Dicer-mediated
processing. In order to ensure inhibition, three dodecapeptides
derived from the amino acid sequence of Dicer and kanamycin (a
known binder of RNA) were tested for modulation of pre-miRNA
maturation. At high concentrations of 100
′
fl uorescein label and a 3
′
M, one peptide was
able to inhibit fl uorescence approximately 85 %, while kanamycin
exhibited a 40 % reduction. This effect was reduced at lower con-
centrations and when using cell lysate Dicer (due to nonspecifi c
binding to cellular nucleic acids) but was still observable at
10-30 %. Based on the initial peptides used in the assay, the
μ
