Experiments to validate the presence and relevance of a microRNA–target pair are laborious and time-consuming. Only a few dozen microRNA–target pairs have been experimentally validated in human, rat, or mouse (see ), despite the prediction of several hundred thousand. The fact that only partial sequence complementarities are needed, however, makes it challenging to ascertain the presence of a microRNA–target pair ( Krutzfeldt et al. This suggests that microRNAs may play an extremely broad and important role in biological regulation. Several thousand protein-coding genes in mammalian genomes have been predicted to be targets of a few hundred known microRNAs. This type of in silico analysis has predicted several hundred thousand microRNA–target pairs in human, rat, and mouse ( John et al. One could predict which genes might be targeted by a microRNA based on the sequence characteristics of known microRNA–target pairs ( Rajewsky 2006). As a result, a microRNA could potentially target multiple genes. Since microRNA exerts its biological effects through suppression of target genes, it is necessary to identify microRNA–target pairs to understand the biological significance of specific microRNAs.Īn interesting characteristic of microRNA is that it only requires partial complementarities with its target sequence. In some cases, microRNA can also reduce the abundance of target mRNA ( Bagga et al. MicroRNA is encoded by specific genes in plant and animal genomes and may act primarily through binding to the 3′ untranslated region of target mRNA and suppressing protein translation ( Ambros 2004 Bartel 2004 He and Hannon 20). MicroRNAs are a class of endogenous, conserved, small regulatory RNA, the discovery of which has been hailed as one of the most important breakthroughs in biology in recent years ( Couzin 2002 Dennis 2002). In addition, this study establishes a differential profile of microRNA expression between the renal cortex and the renal medulla and greatly expands the known differential proteome profiles between the two kidney regions. The identification of reciprocal expression of microRNAs and their computationally predicted targets in the rat kidney provides a unique molecular basis for further exploring the biological role of microRNA. Seven pairs were predicted by two algorithms and two pairs by all three algorithms. Several pairs of reciprocally expressed microRNAs and proteins were predicted to be microRNA–target pairs by TargetScan, PicTar, or miRanda. The differential expression of several microRNAs and proteins was verified by real-time PCR and Western blot analyses, respectively.
From ∼2100 detectable protein spots in two-dimensional gels, we identified 58 proteins as more abundant in the renal cortex and 72 in the renal medulla.
Of the 377 microRNAs analyzed, we identified 6 as enriched in the renal cortex and 11 in the renal medulla. We used microRNA microarray and proteomic techniques to analyze the cortex and the medulla of rat kidneys.
We reasoned that reciprocal expression of a microRNA and a predicted target within a physiological context would support the presence and relevance of a microRNA–target pair.
A major challenge is to obtain experimental evidence for predicted microRNA–target pairs. In silico analysis has predicted that a typical microRNA may regulate the expression of hundreds of target genes, suggesting miRNAs might have broad biological significance. Mammalian genomes contain several hundred highly conserved genes encoding microRNAs.