As an example, it could advantageously be combined with the experimental design recently put forward by Kim and Bartel (2009) in mice

As an example, it could advantageously be combined with the experimental design recently put forward by Kim and Bartel (2009) in mice. miRNA target sites (Chen and Rajewsky 2007); and (4) the observation of downward shifts in the relative transcript levels of the targeted versus untargeted allele in cells of mice heterozygous for SNPs that alter acknowledgement sites for coexpressed miRNAs (Kim and Bartel 2009). At least 10 associations have been reported between complex disease and 3UTR SNPs expected to alter miRNA target sites (Abelson et al. 2005; Zchner et al. 2006; Adams et al. 2007; Mishra et al. 2007; Sethupathy et al. 2007; Tan et al 2007; Beetz et al. 2008; Brendle et al. 2008; Chin et al. 2008; Kapeller et al. 2008; Landi et al. 2008; Lv et al. 2008; Wang et al. 2008; Jensen et al. 2009). However, as BT2 pointed out by Sethupathy and Collins (2008), in most of these instances the evidence assisting the hypothesis was suggestive at best. Moreover, tens of thousands of common SNPs ruin or create putative miRNA target sites in the 3UTR of 12,300 human being genes (e.g., Hiard et al. 2010). Yet, identifying the truly practical target sites, and therefore the relevant SNPs, remains a major challenge. While it seems inescapable that polymorphic miRNA-mediated gene rules makes a significant contribution to phenotypic variance, there is a clear need for approaches that allow effective identification of the related DNA sequence variants. BT2 We herein describe a method that achieves this goal for DNA sequence variants in 3UTRs. It is based on the detection of allelic imbalance in the product of RNA immunoprecipitation (RIP) from cells of heterozygous individuals. We apply the method successfully to the 3UTR mutation of Texel sheep, therefore formally showing its causality and modus operandi. BT2 RESULTS The model: The sheep Texel mutation Quantitative trait loci (QTL) analysis pinpointed a G-to-A transition in the 3UTR of the gene associated with improved muscularity in sheep. The mutation, originating from Texel sheep, was expected to produce an illegitimate 8-mer target site for coexpressed and mRNA was found to be approximately one-third less abundant than wild-type mRNA in skeletal muscle mass of heterozygous animals (compatible with miRNA-dependent target degradation), while circulating levels of MSTN HMGCS1 protein were found to be approximately two-thirds reduced homozygous mutants than in homozygous crazy types (compatible with additional translational inhibition). When launched in the 3UTR of the TK-driven luciferase gene, the substitution caused an gene. Yet, one could argue that the accumulated evidence did not formally show our hypothesis; hence, having to qualify the prospective site as potential in the title (Clop et al. 2006). To provide conclusive evidence in support of our hypothesis, we aimed at demonstrating that transcripts with the mutation more tightly associate with the RNA-induced silencing complex (RISC) than with wild-type transcripts in vivo. To perform the assessment in optimally controlled conditions, we aimed at demonstrating differential RISC association by means of an allelic imbalance test in anti-AGO2 immunoprecipitate from skeletal muscle mass of animals heterozygous for the mutation (Fig. 1). Open in a separate window Number 1. (mutation in the 3UTR of the gene. The approximate positions of the (SM) and (LD) muscle mass examined with this study are demonstrated. (allele on the wild-type with the RISC complex. Prior to immunoprecipitation, transcripts transporting the allele are expected to be slightly more abundant than those transporting the allele in skeletal muscle mass of heterozygous animals as a result of the RISC-dependent degradation of the targeted transcripts. The coimmunoprecipitation of miRNA-regulated mRNA with antibodies directed against RISC parts is expected to cause a online enrichment in targeted transcripts. Luciferase reporter transcripts with four tandem copies of the Texel mutation are preferentially.