In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 236

Fig. 1 Long double-stranded RNAs (dsRNA) and short hairpin RNAs (shRNA) are
processed by Dicer to create small interfering RNAs (siRNA). Pre-miRNAs are cut
by RNAse II-like enzyme Dicer to miRNA duplexes differing from siRNA by the
presence of multiple mismatches. Dicer chops dsRNAs in smaller fragments and
removes the terminal loops in shRNAs and pre-miRNAs

21-23 nucleotide (nt)-long siRNAs actually mediating the gene
silencing [ 4 - 6 ] (Fig. 1 ). DsRNA longer than 30 nt are not suitable
for specifi c gene silencing in mammalian cells because they trigger
nonspecifi c global inhibition of protein synthesis [ 7 - 9 ]. The next
logical step was to try siRNAs themselves (Fig. 1 ), and indeed syn-
thetic siRNAs proved to be effective and specifi c suppressors of the
gene expression in mammalian cells [ 10 , 11 ]. Today, the knock-
down in cultured mammalian cells is most often achieved by trans-
fecting cells with siRNAs. Currently, predesigned siRNAs for many
proteins are available commercially.
Since gene silencing produced by siRNAs in mammals is tran-
sient, an alternative has been to use small hairpin RNAs (shRNA)
in situation when more permanent gene silencing is desired.
ShRNAs (Fig. 1 ) are composed of a short double-stranded stem of
varying length [19-29 base pairs (bp)] and a loop that connects
the strands in the stem forming a hairpin-like structure. These shR-
NAs are encoded by a sequence composed of a sequence for one
strand in the stem followed by the loop sequence and then fol-
lowed by the reversed sequence complementary to the fi rst stem
strand subcloned into an appropriate expression vector. Cells are
transfected with shRNA-encoding expression vector; shRNA is
transcribed in cells, and the single-stranded RNA thus produced
forms hairpin-like secondary structure due to complementary stem
strands. ShRNAs are then processed by the cellular machinery to
yield siRNA. The cellular machinery utilized for shRNA processing
is that of microRNAs (miRNA).

miRNAs are endogenous small noncoding single-stranded RNAs
that regulate gene expression at the posttranscriptional level via
translational repression or degradation of target mRNAs [ 12 - 16 ].
Most animal miRNA are imperfectly complementary to their target
mRNAs recognizing sequences complementary to 7 nt at their

1.1 MicroRNA
Pathway in Mammals