Target mRNA Recognition Research Articles

Retraction: ‘RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA’ (2015), by Cui et al.

Retraction: ‘RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA’ (2015), by Cui <i>et al.</i>

Expression of concern: 'RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA' (2015), by Cui Y et al.

Expression of concern: 'RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA' (2015), by Cui Y et al.

Critical role of backbone coordination in the mRNA recognition by RNA induced silencing complex

Despite its functional importance, the molecular mechanism underlying target mRNA recognition by Argonaute (Ago) remains largely elusive. Based on extensive all-atom molecular dynamics simulations, we constructed quasi-Markov State Model (qMSM) to reveal the dynamics during recognition at position 6-7 in the seed region of human Argonaute 2 (hAgo2). Interestingly, we found that the slowest mode of motion therein is not the gRNA-target base-pairing, but the coordination of the target phosphate groups with a set of positively charged residues of hAgo2. Moreover, the ability of Helix-7 to approach the PIWI and MID domains was found to reduce the effective volume accessible to the target mRNA and therefore facilitate both the backbone coordination and base-pair formation. Further mutant simulations revealed that alanine mutation of the D358 residue on Helix-7 enhanced a trap state to slow down the loading of target mRNA. Similar trap state was also observed when wobble pairs were introduced in g6 and g7, indicating the role of Helix-7 in suppressing non-canonical base-paring. Our study pointed to a general mechanism for mRNA recognition by eukaryotic Agos and demonstrated the promise of qMSM in investigating complex conformational changes of biomolecular systems.

Structural basis for piRNA targeting.

PIWI proteins use PIWI-interacting RNAs (piRNAs) to identify and silence transposable elements and thereby maintain genome integrity between metazoan generations1. The targeting of transposable elements by PIWI has been compared to mRNA target recognition by Argonaute proteins2,3, which use microRNA (miRNA) guides, but the extent to which piRNAs resemble miRNAs is not known. Here we present cryo-electron microscopy structures of a PIWI-piRNA complex from the sponge Ephydatia fluviatilis with and without target RNAs, and a biochemical analysis of target recognition. Mirroring Argonaute, PIWI identifies targets using the piRNA seed region. However, PIWI creates a much weaker seed so that stable target association requires further piRNA-target pairing, making piRNAs less promiscuous than miRNAs. Beyond the seed, the structure of PIWI facilitates piRNA-target pairing in a manner that is tolerant of mismatches, leading to long-lived PIWI-piRNA-target interactions that may accumulate on transposable-element transcripts. PIWI ensures targeting fidelity by physically blocking the propagation of piRNA-target interactions in the absence of faithful seed pairing, and by requiring an extended piRNA-target duplex to reach an endonucleolytically active conformation. PIWI proteins thereby minimize off-targeting cellular mRNAs while defending against evolving genomic threats.

Characteristics of miRNA-SNPs in healthy Japanese subjects and non-small cell lung cancer, colorectal cancer, and soft tissue sarcoma patients.

Single nucleotide polymorphisms in genes encoding microRNAs (miRNA-SNPs) may affect the maturation steps of miRNAs or target mRNA recognition, leading to changes in the expression of target mRNAs to cause gain- or loss-of-function changes. Several miRNA-SNPs are known to be associated with the risk of diseases such as cancer. The purpose of this study was to comprehensively determine the miRNA-SNPs in Japanese individuals to evaluate the differences in allele frequencies between ethnicities by comparing data from the global population in the 1000 Genomes Project and differences between healthy subjects and cancer patients. We performed next-generation sequencing targeting genes encoding 1809 pre-miRNAs. As a result, 403 miRNA-SNPs (146 miRNA-SNPs per subject on average) were identified in 28 healthy Japanese subjects. We observed significant differences in the allele frequencies between ethnicities in 33 of the 403 miRNA-SNPs. The numbers of miRNA-SNPs per subject in 44 non-small cell lung cancer (NSCLC), 33 colorectal cancer (CRC), and 15 soft tissue sarcoma (STS) patients were almost equal to those in healthy subjects. Significant differences in allele frequencies were observed for 14, 11, and 9 miRNA-SNPs in NSCLC, CRC, and STS patients compared with the frequencies in healthy subjects, suggesting that these SNPs can be biomarkers of risk for each type of cancer assessed. In summary, we comprehensively characterized miRNA-SNPs in Japanese individuals and found differences in allele frequencies of several miRNA-SNPs between ethnicities and between healthy subjects and cancer patients. Studies investigating a larger number of subjects should be performed to confirm the potential of miRNA-SNPs as biomarkers of cancer risk.

The Emerging Impact of RNA Editing in Tumor Growth and Metastasis

Context: RNA editing is an essential modification that needs to develop normal cells and is involved in a wide range of biological processes. It can arise in both coding and non-coding sequences with different functional effects. Although the expansion of transcriptome diversity is the primary goal of RNA editing, dysregulation and aberrant editing may act as an essential contributor to cancer pathogenesis. Evidence Acquisition: The current review aimed to investigate the role of RNA editing in cancer initiation and progression. Science Direct and PubMed databases were reviewed from 2000 to 2020 and 2003 to 2020, respectively, using various combinations of "RNA editing" and "cancer" keywords. Results: The location of editing sites has different functional impacts on tumorigenesis. Nonsynonymous editing in antizyme inhibitor 1 (AZIN1) leads to a metastatic progression of colorectal and gastric cancer. Recoding editing events in bladder cancer-associated protein (BLCAP) is correlated with the progression of cervical carcinogenesis. Editing events located at 3′UTRs are also a general mechanism to promote tumor growth in different types of cancers. A significant number of editing events in microRNAs with a functional role in cancer are also reported. These editing sites could change the fate and function of microRNAs, either by preventing target mRNA recognition or by dysregulating an off-target mRNA. Conclusions: There are increasing shreds of evidence on the key role of RNA editing events in cancer initiation and progression.

MRNA structure determines modification by pseudouridine synthase 1.

Pseudouridine (Ψ) is a post-transcriptional RNA modification that alters RNA-RNA and RNA-protein interactions that affect gene expression. mRNA pseudouridylation was recently discovered as a widespread and conserved phenomenon, but the mechanisms responsible for selective, regulated pseudouridylation of specific sequences within mRNAs were unknown. Here, we have revealed new mRNA targets for five pseudouridine synthases and probed the determinants of mRNA target recognition by the predominant mRNA pseudouridylating enzyme, Pus1, by developing high-throughput kinetic analysis of pseudouridylation in vitro. Combining computational prediction and rational mutational analysis revealed an RNA structural motif that is both necessary and sufficient for mRNA pseudouridylation. Applying this structural context information predicted hundreds of additional mRNA targets, that we showed were pseudouridylated in vivo. These results demonstrate a structure-dependent mode of mRNA target recognition by a conserved pseudouridine synthase and implicate modulation of RNA structure as the likely mechanism to regulate mRNA pseudouridylation.

Identification of Arabidopsis CCR4-NOT Complexes with Pumilio RNA-Binding Proteins, APUM5 and APUM2.

CCR4/CAF1 are widely conserved deadenylases in eukaryotes. They form a large complex that includes NOT1 as a scaffold protein and various NOT proteins that are core components of multiple levels of gene expression control. The CCR4-NOT complex also contains several RNA-binding proteins as accessory proteins, which are required for target recognition by CCR4/CAF1 deadenylases. AtCCR4a/b, orthologs of human CCR4 in Arabidopsis, have various physiological effects. AtCCR4 isoforms are likely to have specific target mRNAs related to each physiological effect; however, AtCCR4 does not have RNA-binding capability. Therefore, identifying factors that interact with AtCCR4a/b is indispensable to understand its function as a regulator of gene expression, as well as the target mRNA recognition mechanism. Here, we identified putative components of the AtCCR4-NOT complex using co-immunoprecipitation in combination with mass spectrometry using FLAG-tagged AtCCR4b and subsequent verification with a yeast two-hybrid assay. Interestingly, four of 11 AtCAF1 isoforms interacted with both AtCCR4b and AtNOT1, whereas two isoforms interacted only with AtNOT1 in yeast two-hybrid assays. These results imply that Arabidopsis has multiple CCR4-NOT complexes with various combinations of deadenylases. We also revealed that the RNA-binding protein Arabidopsis Pumilio 5 and 2 interacted with AtCCR4a/b in the cytoplasm with a few foci.

IMP1 KH1 and KH2 domains create a structural platform with unique RNA recognition and re-modelling properties.

IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2–RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets.

Binding of NUFIP2 to Roquin promotes recognition and regulation of ICOS mRNA

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3′-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3′-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3′-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.

MicroRNA-mediated target mRNA cleavage and 3′-uridylation in human cells

MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5′-fragments were 3′-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5′-3′ degradation or other molecular fates. Some 3′-5′ decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA.

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.

Gene expression regulation: lessons from noncoding RNAs.

Gene expression regulation: lessons from noncoding RNAs.

The DEAD Box Helicase RDE-12 Promotes Amplification of RNAi in Cytoplasmic Foci in C. elegans

RNAi is a potent mechanism for downregulating gene expression. Conserved RNAi pathway components are found in animals, plants, fungi, and other eukaryotes. In C. elegans, the RNAi response is greatly amplified by the synthesis of abundant secondary small interfering RNAs (siRNAs). Exogenous double-stranded RNA is processed by Dicer and RDE-1/Argonaute into primary siRNA that guides target mRNA recognition. The RDE-10/RDE-11 complex and the RNA-dependent RNA polymerase RRF-1 then engage the target mRNA for secondary siRNA synthesis. However, the molecular link between primary siRNA production and secondary siRNA synthesis remains largely unknown. Furthermore, it is unclear whether the subcellular sites for target mRNA recognition and degradation coincide with sites where siRNA synthesis and amplification occur. In the C. elegans germline, cytoplasmic P granules at the nuclear pores and perinuclear Mutator foci contribute to target mRNA surveillance and siRNA amplification, respectively. We report that RDE-12, a conserved phenylalanine-glycine (FG) domain-containing DEAD box helicase, localizes in P granules and cytoplasmic foci that are enriched in RSD-6 but are excluded from the Mutator foci. Our results suggest that RDE-12 promotes secondary siRNA synthesis by orchestrating the recruitment of RDE-10 and RRF-1 to primary siRNA-targeted mRNA in distinct cytoplasmic compartments.

Complementarity to an miRNA seed region is sufficient to induce moderate repression of a target transcript in the unicellular green alga Chlamydomonas reinhardtii

MicroRNAs (miRNAs) are 20-24 nt non-coding RNAs that play important regulatory roles in a broad range of eukaryotes by pairing with mRNAs to direct post-transcriptional repression. The mechanistic details of miRNA-mediated post-transcriptional regulation have been well documented in multicellular model organisms. However, this process remains poorly studied in algae such as Chlamydomonas reinhardtii, and specific features of miRNA biogenesis, target mRNA recognition and subsequent silencing are not well understood. In this study, we report on the characterization of a Chlamydomonas miRNA, cre-miR1174.2, which is processed from a near-perfect hairpin RNA. Using Gaussia luciferase (gluc) reporter genes, we have demonstrated that cre-miR1174.2 is functional in Chlamydomonas and capable of triggering site-specific cleavage at the center of a perfectly complementary target sequence. A mismatch tolerance test assay, based on pools of transgenic strains, revealed that target hybridization to nucleotides of the seed region, at the 5' end of an miRNA, was sufficient to induce moderate repression of expression. In contrast, pairing to the 3' region of the miRNA was not critical for silencing. Our results suggest that the base-pairing requirements for small RNA-mediated repression in C. reinhardtii are more similar to those of metazoans compared with the extensive complementarity that is typical of land plants. Individual Chlamydomonas miRNAs may potentially modulate the expression of numerous endogenous targets as a result of these relaxed base-pairing requirements.

Detection of mRNA of the Cyclin D1 Breast Cancer Marker by a Novel Duplex-DNA Probe

Previously, we have described 5-((4-methoxy-phenyl)-trans-vinyl)-2'-deoxy-uridine, 6, as a fluorescent uridine analogue exhibiting a 3000-fold higher quantum yield (Φ 0.12) and maximum emission (478 nm) which is 170 nm red-shifted as compared to uridine. Here, we utilized 6 for preparation of labeled oligodeoxynucleotide (ODN) probes based on MS2 and cyclin D1 (a known breast cancer mRNA marker) sequences. Cyclin D1-derived labeled-ssODN showed a 9.5-fold decrease of quantum yield upon duplex formation. On the basis of this finding, we developed the ds-NIF (nucleoside with intrinsic fluorescence)-probe methodology for detection of cyclin D1 mRNA, by which the fluorescent probe is released upon recognition of target mRNA by the relatively dark NIF-duplex-probe. Indeed, we successfully detected, a ss-deoxynucleic acid (DNA) variant of cyclin D1 mRNA using a dark NIF-labeled duplex-probe, and monitoring the recognition process by fluorescence spectroscopy and gel electrophoresis. Furthermore, we successfully detected cyclin D1 mRNA in RNA extracted from cancerous human cells, using ds-NIF methodology.

Small RNA binding to the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target recognition

The bacterial Sm-like protein Hfq is a central player in the control of bacterial gene expression. Hfq forms complexes with small regulatory RNAs (sRNAs) that use complementary "seed" sequences to target specific mRNAs. Hfq forms hexameric rings, which preferably bind uridine-rich RNA 3' ends on their proximal surface and adenine-rich sequences on their distal surface. However, many reported properties of Hfq/sRNA complexes could not be explained by these RNA binding modes. Here, we use the RybB sRNA to identify the lateral surface of Hfq as a third, independent RNA binding surface. A systematic mutational analysis and competition experiments demonstrate that the lateral sites have a preference for and are sufficient to bind the sRNA "body," including the seed sequence. Furthermore, we detect significant structural rearrangements of the Hfq/sRNA complex upon mRNA target recognition that lead to a release of the seed sequence, or of the entire sRNA molecule in case of an unfavorable 3' end. Consequently, we propose a molecular model for the Hfq/sRNA complex, where the sRNA 3' end is anchored in the proximal site of Hfq, whereas the sRNA body, including the seed sequence, is bound by up to six of the lateral sites. In contrast to previously proposed arrangements, the presented model explains how Hfq can protect large parts of the sRNA body while still allowing a rapid recycling of sRNAs. Furthermore, our model suggests molecular mechanisms for the function of Hfq as an RNA chaperone and for the molecular events that are initiated upon mRNA target recognition.

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

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

The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans

The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

Abstract LB-463: Target mRNA cleavage and degradation mediated by endogenous mammalian microRNAs

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