- Computational Predictions and Experimental Validations of MicroRNAs in Human Adenoviruses
1. Computational predictions
2. Experimental validations (stem-loop RT-PCR, Northern blot, Dot blot and sequencing)
3. miRNA microarray
4. Mass parallel sequencing
People involved: Viet-Hung Trinh
- Global Identification of Potential RNA Targets for the Paralogous La-related RNA Binding Proteins Slf1p and Sro9p
By means of DNA microarrays we aim at determining the RNA targets of yeast RBPs that associate with ribosomes and are thus prime candidates to specifically regulate mRNA translation. Our investigations have focused on two evolutionary conserved La-motif (LM) containing proteins from the yeast Saccharomyces cerevisiae, termed Slf1p and Sro9p. These proteins had previously been shown to bind RNA homopolymers in vitro and to preferentially associate with polysomes (Sobel & Wolin, 1999). We found that the two proteins bind to a largely overlapping set of hundreds of mRNAs. In contrast to the bona fide La protein, which binds to the 3'polyuridine tail of nascent RNA polymerase III transcripts, we could not find significant association of noncoding RNAs with Sro9p and Slf1p. This is in agreement with the fact that Slf1p and Sro9p are mainly cytoplasmic whereas La proteins are primarily localized in the nucleus.
We are currently analyzing and comparing the RNA targets of the two proteins by various means.
People involved: Luca Schenk
- Systematic Analysis of the Crosstalk between Regulatory RNA-Binding Proteins and the MicroRNA Machinery
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.
People involved: Alexander Kanitz, Alessia Galgano, Felix Schnarwiler
- Systematic Identification of RNA-binding Proteins with Protein Microarrays
RNA-binding Proteins (RBPs) are implicated in diverse aspects of post-transcriptional gene regulation. Many RBPs contain characteristic RNA-binding motifs which allowed prediction of hundreds of RBPs in eukaryotic genomes using bioinformatic tools. However, many RBPs may lack known RNA-binding motifs. To identify novel RBPs, we have therefore established a procedure to systematically detect RNA-interacting proteins by applying fluorescently labeled RNA on high-density protein microarrays, which contain more than 4000 proteins from the yeast Saccharomyces cerevisiae. We identified 152 proteins that showed significant and reproducible interaction with mRNA (poly(A) + enriched RNA).
We selected 14 of these RBPs and performed affinity isolations of tagged proteins from yeast cell extracts followed by analysis of associated RNAs with DNA microarrays to identify cellular RNA targets. We found that each of these RBPs was associated with a unique set of RNAs. These preliminary results indicate that many unrecognized RNA-binding proteins exist in eukaryotic proteomes, suggesting novel connections between metabolism and post-transcriptional gene regulation.
Currently, we are using bioinformatics tools, in vitro and functional assays in order to characterize a number of the identified RBPs.
People involved: Tanja Scherrer, Christian Femmer, Oliver Friedli, Alessandra Albertini