Biogenesis of Mammalian miRNA - MicroRNAs as Tools in Biopharmaceutical Production - page 20

2011 ). This review will focus on the role of AGO proteins on the processing and
function of miRNA in mammals. Although the precise mechanism of assembly in
mammals is not yet known, RNA duplex incorporation into AGO proteins and ATP-
dependent RISC assembly appears to be a Dicer -independent process in contrast
to the well-characterized Dicer-dependent RISC assembly involving Ago2 in flies
(Kawamata and Tomari 2010 ; Schwarz et al. 2003 ).
Strand selection by the RISC is a regulated process dependent upon the ther-
modynamic stability of the nucleotides at the 5 termini at each end of the duplex
(Schwarz et al. 2003 ; Khvorova et al. 2003 ). This asymmetric strand selection due to
the relative stability of the 5 end is known as the “asymmetry rule”. The strand with
the least stable 5 end (miRNA or guide strand) is retained within the Ago-associated
complex, whereas the passenger strand is unwound from the duplex and degraded
(Kawamata and Tomari 2010 ; Schwarz et al. 2003 ; Khvorova et al. 2003 ). Recent
deep sequencing studies in Drosophila revealed that miRNA ∗ species for a few
miRNAs are abundantly expressed and dynamically regulated suggesting that
miRNA ∗ might in fact play important gene regulatory roles in a cell-type or cell-stage
specific manner (Okamura et al. 2008 ). Indeed, miRNA ∗ are capable of regulating the
expression of luciferase reporter constructs containing the target sites for miRNA ∗
suggesting that the passenger strands likely play an important role in mammalian
gene regulatory networks (Yang et al. 2011 ).
All four AGO proteins expressed in humans (AGO1-4) can bind miRNA duplexes
containing central mismatches (Yoda et al. 2010 ). In contrast to flies, there does not
appear to be strict small RNA sorting system involved in RISC assembly in mammals.
As a result of mismatches in base-pairing in the duplex, the two strands are unwound
by a cleavage-independent (i.e., nuclease-independent) mechanism (Kawamata et al.
2009 ). Whereas the passenger strand appears to be susceptible to rapid nucleolytic
degradation, the guide strand is retained in the RISC. Upon the formation of a mature
or active RISC, the mature miRNA is then capable of exhibiting a regulatory function
by altering protein translation or mRNA stability.

2.7 MicroRNA Repress Protein Translation

Due to the combinatorial nature of 5 sequence heterogeneity in the seed region of
the miRNA as a result of pri- and pre-miRNA processing, strand selection, and AGO
association, the appropriate target recognition by mature miRNA is essential to its
ability to participate in regulatory networks. Furthermore, miRNA relative expression
levels and subcellular localization contribute to the complexity of miRNA-dependent
regulation of gene expression. Although all four AGO proteins in humans evolved as
ribonucleases, only AGO2 retains its catalytic activity (i.e., Slicer activity) required
for the cleavage of target mRNA (Meister et al. 2004 ). Active RISC containing ei-
ther AGO 1, 3, or 4 loaded with guide miRNA is able to recognize target sequence
in the 3 UTR of target mRNA resulting in translational repression but could also
decrease mRNA stability by promoting transcript degradation (Bartel 2009 ; Eulalio