Biology Reference
In-Depth Information
This 2 nt 3
-overhang of the pre-miRNA is bound by exportin-5
(Exp-5, Ran-binding protein 21), member of the karyopherin fam-
ily of transport proteins and part of a 230 kDa protein complex
which not only protects it from cellular digestion but also mediates
pre-miRNA translocation from the nucleus to the cytoplasm in an
energy-reliant process [
38
-
40
]. During this translocation process
Exp-5 interacts with Ras-related nuclear protein (Ran), a small
25-kDa protein involved in transport in and out of the cell nucleus.
In order that the pre-miRNA is bound by the Exp-5/Ran com-
plex, Ran needs to be in the Ran-guanine triphosphatase (Ran-
GTP) state. The pre-miRNA/Exp-5/Ran-GTP complex is then
translocated to the cytoplasm through a nuclear pore complex.
Once in the cytoplasm, interaction with the Ran-GTPase-activating
protein (RanGAP) facilitates Ran-GTPase activity [
41
]. In conse-
quence of Ran-GTPase activity Exp-5-bound Ran-GTP is hydro-
lized to Ran-guanine diphosphatase (Ran-GDP). The Ran-GTP
hydrolysis leads to pre-miRNA release in the cytoplasm [
11
].
′
3 Extranuclear Maturation: The Cytosolic Precursor-miRNAs and Dicer Cleavage
of Pre-miRNAs to the Mature miRNA
Once the pre-miRNA has been exported to the cytoplasm a second-
round ribonuclease reaction takes place: the RNase III enzyme
Dicer together with its cofactors dsRBD protein Tar RNA-binding
protein (TRBP) and protein activator of PKR (PACT) cleaves the
pre-miRNA into an ~22 nt miRNA/miRNA* duplex with a 2 nt
overhang at each of its 3
end by removing the pre-miRNA hairpin
loop [
42
]. One strand, the mature miRNA guide strand, is loaded
onto Argonaute2 (AGO2) which forms the functional center of an
RNA-induced silencing complex (RISC) [
43
]. The mature miRNA
guide strand guides RISC, the cytoplasmatic effector molecule in
RNA interference (RNAi), to the mRNA target sequence. In
humans, Dicer cleavage activity is regulated via the amino-terminal
DExD/H-box helicase domain which inhibits Dicer activity. TRBP
activates Dicer by binding to this region which results in confor-
mational rearrangement [
44
]. The Dicer product, miRNA let-7,
targets Dicer mRNA, establishing a miRNA/Dicer autoregulatory
negative feedback loop [
45
]. The miRNA-maturing enzyme Dicer,
the dsRBD TRBP, PACT, and Ago2 form a RISC-loading com-
plex (RLC) which facilitates transfer of mature miRNAs from Dicer
to RISC. In some cases when there is a high degree of complemen-
tarity in the hairpin stem of the pre-miRNA, Ago2 generates an
additional processing intermediate, termed Ago2-cleaved precur-
sor miRNA or ac-pre-miRNA [
46
]. This ac-pre-miRNA is nicked
at the miRNA* passenger strand as a result of Ago2-mediated
cleavage of the 3
′
arm of the pre-miRNA hairpin. Dicer is able to
further process the ac-pre-miRNA as other regular pre-miRNAs
[
28
]. Regarding the selection of the miRNA guide strand and its
′
