In Vivo Gene Silencing by Virally Delivered MicroRNA - Viral Vector Approaches in Neurobiology and Brain Diseases - page 237

5 end, usually nt 2-8, known as miRNA “seed” (reviewed in [ 17 ]).

Most miRNAs target the 3

′

-untranslated region of their target
mRNAs, although some are known to target the open reading
frame and even 5

′

-untranslated region, but much less frequently
([ 17 ] and references therein). miRNAs are fi rst produced as pri-
mary transcripts (pri-miRNA) with sequence forming an imperfect
stem of ~33 bp, which contains sequences complementary to the
target mRNA, a single-stranded loop connecting strands in the
stem (a hairpin structure), and adjacent single-stranded 5

′

′ - and

3 -sequences [ 18 - 21 ]. The stem in the hairpin of endogenous miR-

NAs, unlike that of typical shRNAs, is formed of two not quite
complementary strands, thus forming internal loops of variable
length (1-5 nt) [ 18 - 20 ] (Fig. 1 ). The 60-70-nt-long sequence
encoding the miRNA hairpin (pre-miRNA) is excised from pri-
miRNA by endonuclease Drosha in the nucleus [ 21 , 22 ], and pre-
miRNA is exported to the cytoplasm by Exportin-5 [ 23 , 24 ]
(Fig. 2 ). In the cytoplasm, pre-miRNA undergoes further process-
ing. The pre-miRNA has to be cleaved by Dicer, which removes the
terminal hairpin loop producing a ~22-bp-long miRNA duplex
with phosphorylated 5

′

-overhang at each end [ 25 , 26 ],
the structure similar to that of siRNAs but containing mismatches.
The effector complex that is guided by mature miRNA to the target
mRNA is known as RNA-induced silencing complex (RISC) [ 26 ,
27 ]. The RISC contains a member of Argonaute protein family,
among which Argonaute-2 (Ago-2) is the sole member possessing
Mg 2+ -dependent RNA endonuclease activity and able to cleave tar-
get RNA (“Slicer” function) ([ 6 , 13 , 28 , 29 ] see also [ 30 ] and ref-
erences therein). The loading of siRNAs onto RISC is mediated by
RISC-loading complex (RLC). The human RLC contains Dicer as
well as RNAses and dsRNA-binding proteins transactivating
response (TAR) RNA-binding protein (TRBP) and PKR-activating
protein (PACT) that facilitate the Ago-2 recruitment and RISC
assembly [ 31 - 33 ]. In the human, RLC is assembled before the pre-
miRNA processing by Dicer, and pre-miRNA processing and RISC
assembly is functionally coupled [ 33 , 34 ]. Eventually, only one
strand of siRNA or miRNA is retained in the active RISC. Initially,
it was proposed that siRNA duplex is unwound before entering
RISC, with only single-stranded guide strand complementary to
the target mRNA, but not the passenger strand, binding to Ago-2
[ 26 , 27 ]. Recent research suggests that Ago-2 binds siRNA duplexes
and then cleaves the passenger strand [ 35 - 37 ]. It is still unclear
how it is decided during RISC assembly which strand is the right
one (for recent review see [ 38 ]). The guide preferentially incorpo-
rated into RISC has lower stability at the 5

′ - and 2 nt 3 ′

′ end pairs [ 39 , 40 ].

Ago-2 is known to cleave pre-miRNA at 3 end during RISC assem-

bly, which may identify nicked strand as the passenger strand and
facilitate its removal from the mature RISC [ 41 ]. The mature RISC
containing the miRNA guide strand (mature miRNA) bound to

′