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

Aromatic side-chain conformational switch on the surface of the RNA Recognition Motif enables RNA discrimination

The RNA Recognition Motif (RRM) is the most ubiquitous RNA binding domain. Here the authors combined NMR and molecular dynamics simulations and show that the RRM RNA binding surface exists in different states and that a conformational switch of aromatic side-chains fine-tunes sequence specific binding affinities

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

The precursor mRNA (pre-mRNA) retention and splicing (RES) complex is a spliceosomal complex that is present in yeast and humans and is important for RNA splicing and retention of unspliced pre-mRNA. Here, we present the solution NMR structure of the RES core complex from Saccharomyces cerevisiae. Complex formation leads to an intricate folding of three components-Snu17p, Bud13p and Pml1p-that stabilizes the RNA-recognition motif (RRM) fold of Snu17p and increases binding affinity in tertiary interactions between the components by more than 100-fold compared to that in binary interactions. RES interacts with pre-mRNA within the spliceosome, and through the assembly of the RES core complex RNA binding efficiency is increased. The three-dimensional structure of the RES core complex highlights the importance of cooperative folding and binding in the functional organization of the spliceosome