1. Computational predictions
2. Experimental validations (stem-loop RT-PCR, Northern blot, Dot blot and sequencing)
3. miRNA microarray
4. Mass parallel sequencing
It is becoming increasingly clear that the microRNA system and classical post-transcriptional regulatory pathways interact on several levels. In addition to post-transcriptional modifications of the microRNA itself, mRNA binding of regulatory RNA-binding proteins (rRBPs) may influence the efficiency of microRNA-mediated knockdown. Recent studies on the interplay between specific miRNA:mRNA:rRBP pairs suggest the existence of different regulatory mechanisms, such as competition for cis-acting regulatory elements in the 3’ UTR. Moreover, we have previously found that miRNA seeds are enriched in the vicinity of the binding motifs of the rRBPs PUM1 and PUM2.
To gain insight into the nature and extent of this mRNA-dependent crosstalk in human cells, we aim to determine the target sets of several rRBPs on a genome-wide level and compare them with those of the Argonaute proteins (huAGO1-4). In order to achieve this we will employ an improved RBP immunopurification protocol, followed by microarray analysis and/or deep sequencing (‘RIP-Chip’ / ‘RIP-Seq’). Furthermore, interaction networks of the RBPs will be determined by mass spectrometric analysis. Common targets will then be verified and subjected to in-depth bioinformatic and biochemical analysis.
Please see Gerber Lab homepage on OWW for more information.