Role of microRNAs in Regulating Gene Expression

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Segments of the IFN-g 3UTR are conserved across vertebrates and such conserved non-coding sequence (CNS) are hypothesized to have regulatory potential. Our analysis of the IFN-g gene sequence revealed that there is a promising target for miRNAs 29a and b binding downstream of the ARE element in the 3 UTR. This region is present in IFN-g genes cloned from many species, thus implicating this miRNA binding site as having a role in regulating IFN-g gene expression. Typically, both the ARE element and miRNA target sequences are known to be involved in post-transcriptional regulation of gene expression. While in the IFN-g 3 UTR, the ARE is located in the 5 end and the miRNA-29BS at the 3 end, there is no reported evidence supporting the model that miRNAs bind to the ARE region of IFN-g. To test this hypothesis, we generated several 3 IFN-g-UTR reporter constructs by mutating the ARE and miRNA-BS and our experiments revealed competing effects between the ARE and miRNA, affecting the stability of the mRNA. From these results, we hypothesize that the ARE mediated decay (AMD) and RNA induced silencing complex (RISC) pathways competitively interact and thereby regulate post-transcriptional control of IFN-g. We hypothesize that the RISC complex may inhibit the AMD process, by potentially interacting with ARE binding proteins. Our in vitro results led us to enlist the help of Dr. Bruce Shapiro (NCI), an expert in RNA structure. Dr. Shapiros laboratory analyzed the 3 UTR structure and evaluated whether binding of the miRNA altered 3 RNA structure. His laboratory provided computational evidence that binding of mir-29 in the region 1090-1109 leads to a rearrangement of the secondary structure of the mRNA. This structural change appears to be larger and different in nature compared to binding of miRNA in neighboring regions (see Figure 6). In particular, a long-distance interaction between the AU-rich region and the miRNA binding region seems to be modulated by the miRNA binding. The modulation appears to be in the reverse direction as initially expected: the long-distance interaction is predicted to be present upon binding of the miRNA and weakened or not present in the unbound form of the mRNA. However, several caveats of the computational analysis should be mentioned: 1. RNA secondary structure prediction methods that are not based on alignments cannot be expected to be completely accurate. 2. The influence of the bound miRNA on the mRNA secondary structure was estimated with a computational analysis, by subjecting the mRNA sequence to a secondary structure prediction (RNAfold) with the constraint that the nucleotides of the miRNA binding site did not participate in the mRNA folding (remained single stranded) and 3. Structural effects that go beyond RNA base pairing are not modeled. To confirm that the predicted RNA structural changes upon binding of the miRNA actually occur, we initiated a collaboration with Dr. Stuart LeGrice, (NCI) utilizing SHAPE technology (selective 29-hydroxyl acylation analyzed by primer extension). This technology permits us to gain a better understanding of how miRNA binding may affect IFN-g RNA structure and determine if the predicted theoretical changes in RNA structure actually occur following interaction with the miRNAs. If this model is validated, we will then test whether the binding of the protein TTP, already reported to interact with the IFN-g ARE, is altered in the presence of the miRNA. We hypothesize that miRNA binding to the mRNA will interfere with the binding of TTP to the RNA, thus stabilizing the RNA. As part of this overall project, we are also searching for polymorphisms in the IFN-g gene that may effect gene expression by altering IFN-g mRNA stability.