Biogenesis of Mammalian miRNA - MicroRNAs as Tools in Biopharmaceutical Production - page 18

Runx2-mediated increase in miR-2861 and miR-3960 expression plays a key role in
promoting osteoblast differentiation by suppressing inhibitors of Runx2 expression.
Similar to other protein-coding transcripts, these miRNA transcripts known as
primary (pri-) miRNA undergo processing that promotes involving the addition of
a5 -m 7 G cap structure and polyadenylation (Cai et al. 2004 ). These pri-miRNA
transcripts are then processed to a 65-nt precursor (pre-) miRNA in the nu-

cleus by the “Microprocessor” complex consisting of the RNase III-like enzyme
Drosha and its co-factor DiGeorge Syndrome Critical Region 8 (DGCR8; Pasha in
D. melanogaster and C. elegans ) (Lee et al. 2003 ; Denli et al. 2004 ). Pre-miRNA con-
taining hairpin-like structures are exported from the nucleus in a RanGTP-dependent
process involving exportin 5 (Exp5) before undergoing additional cytosolic process-
ing by Dicer to generate a mature miRNA (Bohnsack et al. 2004 ; Lund et al. 2004 ;
Yi et al. 2003 ).

∼

2.3 Nuclear Processing by Drosha

Drosha is a nuclear protein of 130-160 kDa containing two RNase III domains
(RIIID) and a double-stranded RNA binding domain (dsRBD). Mammalian pri-
miRNA transcripts contain dsRNA hairpins consisting of a

∼

33 base pair (bp) stem,
a terminal loop, and 5 and 3 flanking ssRNA. Although Drosha exhibits RNase
III activity in the absence of its co-factor DGCR8, it requires DGCR8 in order to
bind target RNA (Han et al. 2004 ). Instead, Drosha interacts with DGCR8 to form
the core Microprocessor complex that is competent for RNA binding and cleavage.
DGCR8 recognizes the ssRNA-dsRNA junction of target RNA through two dsRBDs
and facilitates substrate cleavage 11 bp from the junction (Han et al. 2006 ; Zeng

and Cullen 2005 ). The two RIIIDs of Drosha cleave the 5

∼

and 3 strands of the

65-nt (60-80-nt) pre-miRNA with a 2-nt 3
overhang (Basyuk et al. 2003 ). The flanking sequence containing the 3 overhang
is an important factor for recognition by EXP5 and subsequent nuclear export for
additional cytosolic processing (Bohnsack et al. 2004 ; Lund et al. 2004 ;Yietal.
2003 ). After binding the pre-miRNA, the EXP5 co-factor GTP-bound nuclear Ran
undergoes cytoplasmic hydrolysis releasing the pre-miRNA into the cytosol.

stem resulting in the formation of a ∼

2.4 Cytosolic Processing by Dicer

Following generation of the pre-miRNA by the Microprocessor complex, the
RNase III-like protein Dicer generates an RNA duplex containing the mature miRNA.
Dicer, an

200 kDa protein containing DEAD-box RNA helicase domain, PAZ do-
main, two RIIIDs, and a dsRBD, cleaves the pre-miRNA near the loop to generate an
∼

∼

22-nt duplex that contains both the mature miRNA (guide strand) and a similarly
sized RNA (passenger strand) derived from the opposite stem of the hairpin (Bern-
stein et al. 2001 ; Grishok et al. 2001 ; Hutvagner et al. 2001 ; Macrae et al. 2006 ).