Forschung

Prof. Dr. phil. W. Eberhardt (group leader)

Contact:

Email: w.eberhardt@em.uni-frankfurt.de

Phone: +49-69 6301-6954 (office)/6966 (lab)

Open positions

Motivated students who wish to conduct a master thesis in Molecular Medicine or medical students aiming to prepare an experimental doctoral thesis are highly welcome to join our laboratory. We offer a close supervision, friendly and most inspiring atmosphere and help candidates to learn standard and modern techniques in Molecular - and Cell Biology.

For further information, please directly contact W. Eberhardt

w.eberhardt@em.uni-frankfurt.de.

Mechanisms of post-transcriptional gene expression by RNA binding proteins in health and disease

Modulation of mRNA turnover and translation are key paradigms of post-transcriptional gene regulation in physiology and pathology of higher eukaryotes. Beside mutations in the genome, dysregulations in gene expression frequently rely on alterations in post-transcriptional control mechanisms. In addition to non-coding RNAs, RNA binding proteins (RBPs) critically determine diverse dynamic mRNA features including splicing, stability, transport, and translation. Emerging evidence from many laboratories demonstrated that alterations in RBP-mRNA interactions can severely disrupt cellular homeostasis and thereby promote inflammatory and tumorigenic processes.

Our group is particularly interested to understand how alterations in RBPs and associated proteins functionally contribute to the pathogenesis of colorectal cancer (CRC) one of the most frequent cancers in humans and among the most prevalent causes of cancer-related deaths worldwide. The mission of our research is the identification of molecular targets which can beneficially be used for novel mechanism-based interference therapeutics e.g., based on small molecule inhibitors, kinase inhibitors as well as diverse RNA interfering strategies.

A special focus of our research is put on the ubiquitously expressed human antigen R (HuR, ELAVL1) a member of the embryonic lethal abnormal vision (ELAV) like proteins and up to now, one of best characterized RBP. In addition to this conventional RBP, in accord with other laboratories, we previously identified the ubiquitin E3 ligase tripartite motif-containing protein (TRIM25) as a novel class of unconventional RBPs characterized by the lack of typical RNA recognition motifs (RRM). Comparable to some other TRIM members, TRIM25 is endowed with RNA binding properties thus highlighting a direct link between protein ubiquitination (frequently but not necessarily associated with the proteasome degradation machinery), and diverse RNA pathways. Functionally, TRIM25 constitutes a class of RNA-binding E3 ligases with a broad pathophysiological relevance e.g. inflammatory diseases and cancer.

Former projects

The E3 Ligase TRIM25 constitutively impairs caspase-2 and caspase-7 through different post-transcriptional mechanisms

• Role of the E3 ubiquitin ligase TRIM25 as a novel post-transcriptional regulator of caspase-2

By employing RNA affinity chromatography in combination with mass spectrometry (MS), we identified TRIM25 as a 5´UTR-caspase-2 mRNA-binding protein. Data from loss-of-function and gain-of-function approaches showed that TRIM25 attenuates the protein levels of caspase-2 without affecting caspase-2 mRNA levels. Functionally, genetic depletion of TRIM25 induced a shift of caspase-2 mRNA from ribonucleoprotein (RNP) particles to translationally active polysomes, indicating that TRIM25 negatively interferes with the translation of caspase-2. The upregulation of caspase-2 in response to TRIM25 silencing was furthermore sensitive to the mechanistic target of rapamycin (mTOR) inhibitor rapamycin thus suggesting that TRIM25 mainly interferes with cap-dependent translation. Functionally, the elevation in caspase-2 upon TRIM25 depletion significantly sensitized colorectal cells to chemotherapeutic drug-induced apoptosis. Inhibition of caspase-2 by TRIM25 therefore implies a survival mechanism that critically contributes to the strong chemotherapeutic drug resistance of colorectal cancer cells.

• Identification of TRIM25 as a novel post-transcriptional regulator of caspase-7 and related cell death

In addition to caspase-2, we identified the apoptosis-related cysteine peptidase caspase-7 as a protein being strongly upregulated in TRIM25 deficient CRC cellls. In clear contrast to caspase-2, we observed an additional increase in caspase-7 mRNA by TRIM25 silencing was accompanied by an elevation in caspase-7 mRNA-stability indicating that TRIM25 interferes with caspase-7 mainly through accelerating decay of caspase-7 encoding mRNA.  Data from RNA pulldown assays with immunoprecipitated TRIM25 truncations identified a direct TRIM25 binding to caspase-7 mRNA, which is mediated by the C-terminal PRY/SPRY domain. This particular domain seems additionally relevant for protein-protein interactions. By employing TRIM25 immunoprecipitation, we identified the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) as a novel TRIM25 binding protein with a functional impact on caspase-7 mRNA stability. Since the interaction of both proteins is highly sensitive to RNase treatment an indirect interaction of both proteins which is mainly achieved through a common RNA binding can be postulated. Results from ubiquitin affinity chromatography indicate that both proteins are targets of ubiquitin modification. Functionally, knockdown of TRIM25 sensitized colon carcinoma cells towards chemotherapeutic-drug-induced apoptosis. Concomitantly, reduced caspase-7 expression in primary colonic tumor samples correlated with a poor survival of patients and concomitantly, a significantly decreased caspase-7 expression was found in primary tumors of CRC patients when compared to adjacent tissue by using the UCSC Xena platform. Our findings suggest that the constitutive suppression of caspase-2 and caspase-7 expression by TRIM25 through interference with different post-transcriptional control mechanisms (translation vs. mRNA-stability) constitutes a novel survival mechanism of colon carcinoma cells which additionally contributes to chemotherapeutic drug resistance.