miRNA repression involves GW182-mediated recruitment of CCR4-NOT through conserved W-containing motifs.

miRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR4-NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR4-NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR4-NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNAs, including inhibiting translation independently of deadenylation.

An investigation of the structural requirements for ATP hydrolysis and DNA cleavage by the EcoKI Type I DNA restriction and modification enzyme.

Type I DNA restriction/modification systems are oligomeric enzymes capable of switching between a methyltransferase function on hemimethylated host DNA and an endonuclease function on unmethylated foreign DNA. They have long been believed to not turnover as endonucleases with the enzyme becoming inactive after cleavage. Cleavage is preceded and followed by extensive ATP hydrolysis and DNA translocation. A role for dissociation of subunits to allow their reuse has been proposed for the EcoR124I enzyme. The EcoKI enzyme is a stable assembly in the absence of DNA, so recycling was thought impossible. Here, we demonstrate that EcoKI becomes unstable on long unmethylated DNA; reuse of the methyltransferase subunits is possible so that restriction proceeds until the restriction subunits have been depleted. We observed that RecBCD exonuclease halts restriction and does not assist recycling. We examined the DNA structure required to initiate ATP hydrolysis by EcoKI and find that a 21-bp duplex with single-stranded extensions of 12 bases on either side of the target sequence is sufficient to support hydrolysis. Lastly, we discuss whether turnover is an evolutionary requirement for restriction, show that the ATP hydrolysis is not deleterious to the host cell and discuss how foreign DNA occasionally becomes fully methylated by these systems.

Mammalian miRNA RISC recruits CAF1 and PABP to affect PABP-dependent deadenylation.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.

Importance of the C-terminal domain of the human GW182 protein TNRC6C for translational repression.

Proteins of the GW182 family play an important role in the execution of microRNA repression in metazoa. They interact directly with Argonaute proteins, components of microRNPs, and also form part of P-bodies, structures implicated in translational repression and mRNA degradation. Recent results demonstrated that Drosophila GW182 has the potential to both repress translation and accelerate mRNA deadenylation and decay. In contrast to a single GW182 protein in Drosophila, the three GW182 paralogs TNRC6A, TNRC6B, and TNRC6C are encoded in mammalian genomes. In this study, we provide evidence that TNRC6C, like TNRC6A and TNRC6B, is important for efficient miRNA repression. We further demonstrate that tethering of each of the human TNRC6 proteins to a reporter mRNA has a dramatic inhibitory effect on protein synthesis. The repression is due to a combination of effects on the mRNA level and mRNA translation. Through deletion and mutagenesis, we identified the C-terminal part of TNRC6C encompassing the RRM RNA-binding motif as a key effector domain mediating protein synthesis repression by TNRC6C.