Biomedical Engineering Reference
In-Depth Information
positions 2 and 8 (seed sequences/region) must be perfectly base-paired to start an
interaction between miRNP and mRNA (Brennecke et al., 2005; Doench and
Sharp, 2004; Grimson et al., 2007; Lewis et al., 2005; Nielsen et al., 2007).
2 Mechanisms of miRNA Function
miRNAs regulate the translation or degradation of target mRNAs by forming a
partial hybrid duplex at their cognate target sequences in the 3
0
UTRs of target
mRNAs. There are two different ways miRNAs can influence gene expression
as discussed below (Fig. 1).
2.1 Translational Repression
There is now evidence suggesting that miRNP-mediated repression of transla-
tion might occur both before and after the initiation of translation.
2.1.1 Pre-initiation Repression of Translation
The presence of the m
7
G cap at the 5
0
end of mRNAs is essential for miRNA-
mediated repression of translation initiation (Humphreys et al., 2005; Pillai et al.,
2005). This requirement was further proven by bi-cistronic reporter assays and
polysome gradient analyses. In bi-cistronic reporter assays (where one cistron
was placed under cap and the other under IRES (internal ribosome entry site)), it
was shown that only the cap-dependent cistron was specifically repressed. In
polysome gradient experiments, it was shown that the reporter mRNAs with
miRNA target sequences or artificially tetheredAGO2 protein at the 3
0
UTR had
low sedimentation coefficients, suggesting a lack of translational initiation. In
another mechanism, the AGO2 protein plays a central role, where it has been
proposed to compete for m
7
G cap with eI4E (a cap-recognizing translation
initiation factor). The central domain of AGO2 protein has sequence similarities
with eI4E, and when mutated AGO2 is unable to mediate translational repres-
sion,evenwhenartificiallytetheredtothe3
0
UTR sequences (Kiriakidou et al.,
2007). This observation may further explain why the presence of multiple
miRNA-binding sites in the 3
0
UTR is able tomediate a robust miRNA-mediated
repression of translation. It is conceivable that the presence of multiple low-cap-
affinity AGO2 proteins at the 3
0
UTR is a more efficient inhibitor of eI4E binding
at the cap. It was further established that mRNAs to be targeted by miRNA-
mediated inhibition of translation should also have a poly(A) tail (Wakiyama
et al., 2007). The inhibition of assembly of 60S and 40S ribosomal subunits has
also been proposed as a mechanism of miRNA-mediated translation inhibition.
It was shown that the AGO2-Dicer-TRBP complex interacts with eIF6 and 60S
ribosomal subunits. Partial depletion of eIF6 in cells leads to rescue of miRNA-
mediated repression of target mRNAs (Chendrimada et al., 2007).
