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.
Conserved CpG-island Promoters Drive Transcription of Telomeric Repeat-Containing RNA in Human Cells[show][hide]
Non-Hodgkin lymphomas (NHL) comprise a very heterogeneous group of human malignant diseases with thus far not fully understood pathogenesis. NHLs can be further sub-divided into entities of B-Cell or T-Cell origin of which the B-Cell Lymphoma proportion accounts for approximately 80%. Oncogenic virus like the Ebstein-Barr-Virus (EBV) is considered as one major cause of malignant B-Cell transformation in vivo. EBV strongly associated with lymphomas occurring in immuno-compromised but as well in competent individuals. Frequency varies between 10-35% in diffuse large B-Cell Lymphomas and >95% in Burkitt-Lymphoma. Beside other factors like genomic aberrations within the cellular context, several latently expressed viral antigens like EBNA-2 and LMP1 seem to be critical for transforming potential of EBV. Besides that, EBV has been found to express up to different 26 microRNAs independently EBV latency states in situ. Functional significance for most of these EBV encoded miRNAs especially for tumorigenesis remains to be elucidated. Because EBV is well adopted to its host, it is also reasonable that this virus is able to manipulate overall cellular miRNA expression pattern for replication and transformation purposes. Nevertheless, recent studies show that certain miRNA species have capacity for diagnosis and prognosis purposes of special B-Lymphoma sub-sets, but the particular impact of EBV on global miRNA profile NHL B-Cell lymphoma development has not yet deeply examined. The aim of the present study is therefore to evaluate the contribution of EBV to global miRNA expression, lymphomagenesis and tumor progression.
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]
Members of the conserved PUF (Pumilio and FBF) protein family selectively bind the 3'UTR of their target mRNAs to regulate translation and thereby exert spatiotemporal control on various developmental processes, such as proliferation and self-renewal of stem cells. In the C. elegans germline, the PUF proteins FBF-1 and FBF-2 maintain germline stem cells by restricting the activity of the differentiation- and apoptosis promoting MAP-Kinase (MAPK). FBF-1, FBF-2 and PUF-8 also act in the soma to influence cell fate decisions in the C. elegans vulva.
During vulval development, cell fates are specified by differential activation of the RAS/MAPK pathway in adjacent vulval precursor cells (VPCs). The cell with the strongest RAS/MAPK activation adopts the primary cell fate and induces the lateral inhibition signal (LIN-12/Notch) in the neighbouring VPCs to lower MAPK signalling and promote the secondary cell fate. PUF-8 was shown to restrict the competence of VPCs to respond to inductive signals, whereas the FBFs restrain the RAS/MAPK signal to ensure that only one cell adopts the primary cell fate (Walser et al., 2006).
Although the consensus binding sites for PUF proteins have been determined, the physiological mRNA targets of the FBFs and PUF-8 in distinct cells are to date unknown. In order to elucidate the different mechanisms by which individual PUF proteins influence cell fate decisions, we are employing an RNA binding protein immunopurification protocol by expressing PUF proteins fused to an HA-Strep-tag III in worms. mRNA targets will then be determined by microarray analysis of co-purified RNA (RIP-Chip), and interaction partners of the PUF proteins will be identified by mass spectrometric analysis. Finally, putative targets and interaction partners will be extensively analysed by genetic, biochemical and bioinformatics means.
Single Molecule Folding Studies of Group II Intron Ribozymes[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]