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Abstract:
Animal miRNAs silence the expression of mRNA targets
through translational repression, deadenylation and subsequent
mRNA degradation. Silencing requires association of miRNAs
with an Argonaute protein (AGO) and a GW182 family protein.
In turn, GW182 proteins interact with the PAN2-PAN3 and
CCR4-NOT deadenylase complexes. These interactions are
required for silencing of miRNA targets. GW182 proteins are
characterized by tryptophan (W)-containing motifs, which have
been shown to mediate the interactions with the Argonaute pro-
teins, and the PAN2-PAN3 and CCR4-NOT deadenylase com-
plexes. In molecular terms, it has been speculated that the
tryptophan residues are accommodated in hydrophobic pockets
of the protein partners and that several such pockets and their
spatial arrangement could confer increased affinity and specific-
ity. We are combining cellular, biochemical and structural
approaches to investigate how the GW182 proteins interact with
their partners to mediate silencing. Our studies uncovered the
presence of W-binding pockets in PAN3 and the CNOT9 subunit
of the CCR4-NOT complex, revealing the structural basis for the
recruitment of deadenylase complexes to miRNA targets. We fur-
ther demonstrated that a central domain in the CNOT1 subunit
of the CCR4-NOT complex interacts with the CAF1 and CCR4
deadenylases and with the RNA helicase DDX6. DDX6 is a
translational repressor and decapping activator, which has been
implicated in the miRNA pathway. Together, our data provide
the missing physical links in a molecular pathway that connects
miRNA target recognition with translational repression, deadeny-
lation and decapping.
