Abstract LB-464: Gene silencing by the mammalian microRNA-mediated and the mRNA-structure-dependent target mRNA cleavage, uridylation, and degradation

Abstract

Abstract Argonaute (Ago) family proteins and GW182 are key components in microRNA(miRNA)-induced silencing complex (miRISC) and have recently been shown to play an important role in miRNA-mediated target gene expression interference by facilitating mRNA deadenylation, destabilization, and translational repression. However, the precise mechanism in mammalian miRNA-mediated mRNA degradation remains unknown. In this study, we used a stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) to specifically and efficiently detect the 3′ termini of cleaved mRNA fragments targeted by endogenous miR-98 in 3′-untranslated region (3′UTR) of a tumor suppressor gene TUSC2 mRNA. We detected the accumulation of miR-98 cleaved 5′ fragments of TUSC2 mRNA in its native forms by native SLA-RT-PCR and 3′-uridinylated forms by U-tract-specific SLA-RT-PCR, respectively, around miR-98 target sites. Significantly elevated bands with U-tract additions compared to the native ones of the cleaved target mRNA 5′ fragments were detected in mRNA sequences corresponding to the miR-98 seed region, 3′-complimentary pairing region, and as well as the regions immediately adjacent to the 3′- and 5′- ends of miR-98 sites, but no accumulation was detected in the central budge region of miR-98 in total RNAs isolated from human lung cancer H1299 cells. Knocking down terminal uridyltransferase 3 (TUTase-3) and TUTase-2 by gene-specific siRNAs respectively diminished and significantly reduced the accumulation of U-tract added fragments, confirming the miR-98-mediated target mRNA cleavage and 3-uridylation. We constructed an EGFP reporter plasmid vector by inserting two identical copies of TUSC2 miR-98 target sequences with 200 bp apart at the 3′ UTR of EGFP reporter gene. We transiently transfected this plasmid into human lung cancer H1299 cells with a high endogenous miR-98 expression or co-transfected with a miR-98-expressing plasmid into mouse F9 cells in total RNAs isolated at designed time points to analyze the potential effects of secondary or 3-dimensional (3D) structures of target mRNA on the precise positions and efficiency of the dynamic miR-98-mediated target mRNA cleavage and 3-uridylation in living cells. We found that the patterns, positions, and effectiveness of miRNA-mediated target mRNA cleavage and 3′-uridylation are significantly influenced by the unique secondary or 3D structures of target mRNAs. Our findings provided new insight into molecular mechanism in mammalian miRNA-mediated gene silencing by a cooperative action in miRISC through the miRNA-mediated and mRNA-structure-dependent target mRNA recognition, cleavage, uridylation, and degradation in mammalian cells. (Supported by grants P50CA70907, RO1 CA-116322, and W81XWH0920139). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-464. doi:1538-7445.AM2012-LB-464