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Abstract

The nonsense-mediated mRNA decay (NMD) pathway triggers rapid degradation of aberrant mRNAs that contain
premature translation termination codons (PTCs). In metazoans, NMD requires three 14-3-3-like proteins: SMG5,
SMG6, and SMG7. These proteins are recruited to PTC-containing mRNAs through the interaction of their 14-3-3-like
domains with phosphorylated UPF1, the central NMD effector. The recruitment of SMG5, SMG6, and SMG7 causes
NMD target degradation. SMG6 possesses an active PIN domain at its C-terminus that cleaves the target mRNA in
the vicinity of the PTC. On the other hand, tethered SMG7 has been shown to degrade mRNA efficiently through its
Proline-rich C-terminus (PC) region, which is necessary and sufficient for this activity. Previous studies indicate that
SMG7-mediated mRNA degradation requires the general mRNA decay enzymes. However, the mechanism by which
these enzymes are recruited to the target mRNA has remained unclear. To determine how SMG7 elicits mRNA decay,
we used the Tandem-Affinity Purification (TAP-tag) methodology to identify interacting partners. Amongst the SMG7-
binding proteins, we found all subunits of the CCR4-NOT complex that are responsible for deadenylation of mRNAs.
We further show that the PC-region of SMG7 is responsible for binding POP2, the catalytically active component of
the CCR4-NOT complex. Additional mapping experiments of POP2 reveal that the catalytic domain alone is sufficient
to bind the PC region of SMG7. Over-expression of catalytically inactive POP2 shows stabilization of SMG7-tethered
RNA and PTC-containing reporter RNA. In addition, over-expression of a catalytically inactive DCP2, the decapping
enzyme, also results in stabilization of NMD targets. Functional studies in human cells demonstrate that the PC region
of SMG7 is required for NMD only in cells depleted of SMG6. Together with previously published data, our findings
show that the 5’ to 3’ decay pathway is utilized in the SMG7-dependent degradation of NMD targets.