Derepression of MicroRNA-mediated Protein Translation Inhibition by Apolipoprotein B mRNA-editing Enzyme Catalytic Polypeptide-like 3G (APOBEC3G) and Its Family Members

Abstract

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNAbinding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies. MicroRNAs (miRNAs)2 are 20 -22-nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages, including plants, insects, vertebrate, and mammals (1-3). More than 474 miRNAs have been identified in humans so far, and ϳ30% of the genes in the human genome are predicted to be subject to miRNA regulation (4). The expression of many miRNAs is usually specific to a tissue or developmental stage, and the miRNA expression pattern is altered during the development of many diseases (3). Mature miRNAs are generated from RNA polymerase II-transcribed primary miRNAs that are processed sequentially by the nucleases Drosha and Dicer. Although miRNA can guide mRNA cleavage, the basic function of miRNA is to mediate inhibition of protein translation (1, 5-8) through miRNA-induced silencing complexes (miRISCs). The guiding strand of miRNA in a miRISC interacts with a complementary sequence in the 3Ј-untranslated region (3Ј-UTR) of its target mRNA by partial sequence complementarities, resulting in translational inhibition (1). A 7-nucleotide "seed" sequence (at positions 2-8 from the 5Ј-end) in miRNAs seems to be essential for this action (4). The composition of the miRISC is similar to that of the RNA-induced silencing complex (RISC), which is responsible for mRNA cleavage guided by small interfering RNAs (siRNAs) (1, 3, 7). Nevertheless, some differences exist between miRISCs and siRNA RISCs. For example, the major Argonaute protein in siRNA RISC is Ago-2, whereas all four of the Ago proteins (Ago1-4) are found in miRISC (3,8). Further, the siRNA RISC may be associated with various RNA-binding proteins such as fragile-X mental retardation protein (FMRP), TAR RNA-binding protein (TRBP), and the human homolog of the Drosophila helicase Arm...

APOBEC3G Inhibits MicroRNA-mediated Repression of Translation by Interfering with the Interaction between Argonaute-2 and MOV10

“…We were especially interested in MOV10, which has been described as a novel component of Argonaute-associated RNP complex (9). Because A3G is also the Argonaute-associated protein and can counteract miRNA-mediated protein repression, we studied the interaction between A3G and MOV10 (11,12). As shown in supplemental Fig.…”

“…A3G Counteracts miRNA-mediated Gene Silencing by Inhibiting the Interaction between MOV10 and AGO2-Our previous study has shown that A3G can counteract miRNA-mediated protein inhibition (12). To determine the details of the molecular mechanism, we placed one copy of mir25-and mir16-binding sites with either perfectly or partially complementary to their corresponding miRNAs into the 3Ј-UTR of the reporter gene.…”

“…Plasmids-Wild-type and mutant plasmids expressing A3G were constructed as described previously (12,15). Human MOV10 with FLAG or HA epitope tag sequences at their 3Ј termini were amplified through reverse transcription-polymerase chain reaction (RT-PCR) with the mRNA of 293T cells as the template.…”

“…These associations are "bridged" by RNAs. Because these proteins are components of the miRISC, A3G can be involved in miRNA-related activities (11,12,14). We have previously demonstrated that A3G and other APOBEC3 family members can counteract the inhibition of protein translation by various miRNAs (12).…”

“…Because these proteins are components of the miRISC, A3G can be involved in miRNA-related activities (11,12,14). We have previously demonstrated that A3G and other APOBEC3 family members can counteract the inhibition of protein translation by various miRNAs (12). Through various approaches, we here demonstrate that A3G counteracts miRNA-mediated gene silencing by competitively inhibiting the interactions between MOV10 and AGO2.…”