Malignant melanoma represents the most lethal cutaneous neoplasm and has reached epidemic proportions for most industrialized countries. Responsive rates to conventional treatment approaches have yet remained largely unsatisfactory once the tumor has started to metastasize.
Different strategies have been developed aiming at gene therapy for diseases including monogenic disorders or cardiovascular diseases, but mainly for the treatment of cancer. Most attempts were hampered by the lack of an efficient gene delivery system. Virotherapy using conditionally replicating adenoviruses (CRAds) exploits their oncolytic potential combined with specificity for cancer cells, while somatic tissues remain unharmed.
Malignant melanoma was previously addressed by generating the CRAd Ad∆EP-TETP that would allow high levels of virus replication and specificity for the tumour tissue, by substituting the promoter of E1A (major transactivator of adenoviral replication) and its enhancer elements by a composite promoter tyrosinase-dependant enhancer/promoter. We propose to foster this approach by restricting other viral genes introducing another cancer-specific promoter. The proposed further improvement of our melanoma-specific CRAd by additional targeting strategies and simultaneous expression of diverse therapeutic proteins may enhance the development of new therapeutic approaches using oncolytic Ads.
Global Identification of Potential RNA Targets for the Paralogous La-related RNA Binding Proteins Slf1p and Sro9p[show][hide]
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.
Identification and Validation of Potential miRNAs Encoded by Human Adenoviruses[show][hide]
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.
Systematic Identification of RNA-binding Proteins with Protein Microarrays[show][hide]
People involved: Tanja Scherrer, Christian Femmer, Oliver Friedli, Alessandra Albertini (Gerber Lab)
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.
The role of microRNAs in the regulation of fetal hemoglobin[show][hide]