Abstract LB-463: Target mRNA cleavage and degradation mediated by endogenous mammalian microRNAs
Authors: Jing Lin, Kai Xu, Jack A. Roth, Lin Ji
Journal: Cancer Research (2012)

Abstract Recent studies suggest that the mammalian microRNAs (miRNAs) predominately act to decrease target mRNA levels, leading to reduced protein output. However, no direct evidence for miRNA-mediated target mRNA cleavage and degradation has been demonstrated in mammalian cells, in which such efforts are hampered by lacks of tools for accurately and reliably detecting the miRNA target cleavage sites and the cleaved mRNA products. In this study, we developed a sensitive, efficient, and the cleavage site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) technique and used the established miR-98-targeted high mobility group A2 (HMGA2) mRNA as a model system to determine the potential miRNA-directed mRNA target recognition, cleavage, and degradation in context of UTRs in mammalian cells under physiological conditions. We detected cleavage activities among the eight computer-predicted potential miR-98 target sites in UTRs of HMGA2 mRNA with varied efficiency, as demonstrated by the unique and specific SLA-RT-PCR products and patterns by agarose gel electrophoresis and confirmed by DNA sequencing. The first predicted miR-98 target site in HMGA2 3′ UTR is within 15 nt from the stop codon and showed almost no cleavage activity. The last two miR-98 target sites near the 3′ end of HMGA2 mRNA showed a significantly less cleavage efficiency than those in their up-stream 3′UTR target sites. These results suggest that the microRNA induced silencing complex (miRISC) might utilize a 5′ to 3′ mRNA scanning model similar to the translation machinery for processing mRNA target site recognition and cleavage and that the miRNA-directed target cleavage activity and efficiency are influenced by the positional and the structural contexts in the 3′UTR of the target mRNA. We also found that upon miRNA/Argonaute 2 (Ago 2) -mediated target mRNA cleavage a tract of uridine bases (U-tract) was added onto the exposed 3′ end of the resulting 5′ fragment, as demonstrated by an increased intensity of the cleaved fragments by SLA-RT-PCR with U-tract-specific detecting primers. The 3′-uridylation of the cleaved mRNA may lead to a 5′ to 3′ directional exonucleolytic decay, as evidenced by the undetectable 3′ to 5′ decay fragments in all miR-98 target sites on HMGA2 mRNA by SLA-RT-PCR. Furthermore, we also detected the miR-98-mediated cleavage at a 5′UTR target site of HMGA2 mRNA although the cleavage activity and efficiency are weaker and lower than those observed in 3′ UTR, providing an additional mechanism to the conventional model that restricts miRNA targets to the 3′UTR. Our findings provide direct evidence and new insights into molecular mechanism on mammalian miRNA-mediated gene expression repression by targeting mRNA cleavage and degradation. (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-463. doi:1538-7445.AM2012-LB-463