A molecular link between miRISCs and deadenylases provides new insight into the mechanism of gene silencing by microRNAs

Abstract

MicroRNAs (miRNAs) are a large family of endogenous noncoding RNAs that, together with the Argonaute family of proteins (AGOs), silence the expression of complementary mRNA targets posttranscriptionally. Perfectly complementary targets are cleaved within the base-paired region by catalytically active AGOs. In the case of partially complementary targets, however, AGOs are insufficient for silencing and need to recruit a protein of the GW182 family. GW182 proteins induce translational repression, mRNA deadenylation and exonucleolytic target degradation. Recent work has revealed a direct molecular link between GW182 proteins and cellular deadenylase complexes. These findings shed light on how miRNAs bring about target mRNA degradation and promise to further our understanding of the mechanism of miRNA-mediated repression.

Figure 1.

Mechanism of miRNA-mediated gene silencing (fully or nearly complementary targets). A miRNA bound to an AGO protein recognizes mRNA targets containing fully or nearly complementary binding sites. In plants, these binding sites are predominantly located within the ORF. The AGO protein cleaves the mRNA within the base-paired region (between nucleotides opposite to nucleotides 10 and 11 of the miRNA strand, which should be base-paired for AGO to cleave). Following cleavage, the mRNA decay intermediates are degraded from the newly generated 3′ and 5′ ends by the exosome and XRN1 or its plant ortholog XRN4 (not shown; Voinnet 2009). The miRNA 5′ terminal nucleotide (shown in black) is buried in the 5′-phosphate binding pocket of AGOs and is not available for pairing with the target.

Figure 2.

miRNA target recognition in animals. In animals, miRNAs typically recognize partially complementary binding sites, which are generally located in 3′ UTRs. Complementarity to the miRNA “seed” sequence, containing nucleotides 2–7 or 2–8, is a major determinant in target recognition and is sufficient to trigger silencing. In some cases, complementarity to the 3′ region of the miRNA might contribute to target binding (not shown, see Bartel 2009). However, even for these sites, miRNA nucleotides 9–12 are generally not complementary to the target, preventing endonucleolytic cleavage by AGOs. In these cases, AGOs recruit a protein of the GW182 family (see Fig. 3).

Figure 3.

Mechanism of miRNA-mediated gene silencing in animals (partially complementary targets). (A,B) The AGO–GW182 complex represses translation through an unknown mechanism and directs the mRNA to deadenylation. Human GW182 proteins interact with PABPC through the PAM2 motif, with PAN3 through the M2 and carboxy-terminal (C-term) regions and with NOT1 through the M1, M2, and C-term regions. Although translational repression and deadenylation are shown as consecutive steps, the order of events remains controversial, and it is unclear whether the two processes are linked or independent. Depending on the cell type and/or specific target, deadenylated mRNAs can be stored in a deadenylated, translationally repressed state. In animal cell cultures, deadenylated mRNAs are generally decapped and rapidly degraded by the major 5′-to-3′ exonuclease XRN1.

Figure 4.

GW182 protein family. Domain organization of GW182 proteins. The amino-terminal AGO-binding domains contain multiple GW repeats (not shown). The silencing domain includes the Mid and carboxy-terminal regions but not the RRM, which is dispensable for silencing. The Mid and C-term regions contain a variable number of GW repeats (not shown) and additional motifs termed CIM-1, CIM-2, and P-GL. Human TNRC6C and Dm GW182 are shown as representative family members of the GW182 proteins. Abbreviations: UBA, ubiquitin-associated domain; Q-rich, region rich in glutamine; Mid, middle domain with PAM2 motif (dark blue), which divides the Mid region into the M1 and M2 regions; RRM, RNA recognition motif; C-term, carboxy-terminal region; N-term, amino-terminal.