RNA-dependent Protein Complexes

The function of RNAs - both coding mRNAs as well as non-coding RNAs (ncRNAs) - is intricately linked to their ability to interact with DNA, RNA or proteins. Already in the early days of lncRNA research, we hance developed a string interest in Ribonucleoprotein (RNP) complexes and RNA-binding proteins in cancer.

We uncovered the important oncogenic function of IGF2BP1 in liver cancer (Hepatology 2014) as well as interaction partners of the mRNA of the immune activating ligand MICB (Nat Commun 2014).
We also established state-of-the-art techniques in our lab for the identification of RNA-protein complexes in vitro and in vivo (Nature 2015).

Currently, we are most excited about a new concept developed in our lab: RNA dependence (Mol Cell 2019). We define a protein as RNA-dependent if its interactome depends on RNA. This functional definition has a lot of important implications overcoming the questions of specificity and quantitation of the common definition of RNA-binding proteins. We have recently performed a successful proteome-wide screen defining RNA-dependent proteins - and make it available to the scientific community in our R-DeeP database.

70% of the human genome are transcribed - but only 2% encode for proteins!

Long Non-coding RNAs in Cancer

One major field of our research aims at the elucidation of the molecular and cellular function as well as expression profiles and regulatory mechanisms of long non-coding RNAs (lncRNA) in cancer. Recent studies suggest that a large part (up to 70%) of the human genome is transcribed, while only 2% of the human genome encodes for proteins. However, for most of the ncRNA transcripts, their functions remain to be discovered. Interestingly, many of these ncRNAs are differentially expressed in cancer compared to normal tissue and hence could serve as biomarkers or therapeutic targets in the future. Additionally, many ncRNAs are highly conserved during evolution which provides another indication of potential functional importance of this diverse class of molecules.

Initially, we carried out microarray-based profiling of 17000 ncRNAs in three major tumor entities: lung, liver and breast cancer. This analysis has identified hundreds of differentially expressed non-coding RNAs specifically expressed or silenced in human cancer.
Our research focuses on differentially expressed ncRNAs in lung adenocarcinoma, hepatocellular carcinoma and mammary carcinoma. For these, we determine the cellular functions by studying gain- and loss-of-function models. To reveal the molecular function of these novel long non-coding RNAs, we use RNA affinity purifications to elucidate the ncRNA-protein networks (Nature 2015).

As an important novel technique for studying loss-of function phenotypes in human tumors, we developed a method to knockout ncRNAs in human cancer cell lines using Zinc Finger Nucleases (Genome Res 2011). Today, we are using the CRISPR / Cas9 system for gain- and loss-of-function models of lncRNAs - however, we also realize the additional challenges and the required measures of caution when working with lncRNAs in complex loci (Nucleic Acids Res 2017).

The prime example of our research is the study of the lncRNA MALAT1: Sven Diederichs already discovered MALAT1 during his PhD thesis as a marker associated with metastasis development in lung cancer (Oncogene 2003). We then established a loss-of-function model for MALAT1 in human lung cancer cells (Genome Res 2011). This enabled us to demonstrate that MALAT1 was not only a marker, but an active and essential player in lung cancer metastasis. It could serve as a therapeutic target for metastasis prevention therapy. It functioned as an epigenetic regulator of a pro-migratory, pro-invasive gene signature (Cancer Res 2013). Surprisingly and despite the strong impact on lung cancer cell migration, knockout of MALAT1 in the mouse did not result in any observable phenoptype (RNA Biol 2012).

Additionally, we unraveled the regulation, function and mechanism of additional lncRNAs like HULC (Hepatology 2013), LIMT (EMBO Mol Med 2016), TP53TG1 (PNAS 2016), VELUCT (Nucleic Acids Res 2017), linc00152 (Sci Rep 2017) and NOP14-AS1 (Nucleic Acids Res 2017).

To study lncRNA function using high-throughput technologies, we developed and screened our own custom-designed siRNA library targeting 638 lncRNAs upregulated in lung, liver and breast cancer (Nucleic Acids Res 2017, Sci Rep 2017). To further extend the spectrum, we have now designed and generated a library for CRISPR / Cas9-based screens targeting 2100 genes (mRNAs and lncRNAs) linked to lung adenocarcinoma by 42000 sgRNAs. These screens will elucidate novel pathways, mechanisms, vulnerabilities and synthetic lethalities in this deadly disease.

Model of miRNA Biogenesis

Model of miRNA Biogenesis

MicroRNA Biogenesis & Regulation

Priot to the aforementioned projects, we focused on the biogenesis, processing and regulation of microRNAs. microRNAs are short RNA molecules, that regulate gene expression on a post-transcriptional level by either destabilizing mRNA molecules or by inhibiting their translation. They are generated in a multistep pathway from longer precursors including multiple RNase cleavage reactions (Nature Cell Biology, 2009). The primary transcript, the pri-miRNA, is cleaved by the nuclear RNase Drosha into the shorter hairpin, the precursor microRNA or pre-miRNA, which is exported from the nucleus by the Exportin-5 / Ran-GTP complex. The cytoplasmic pre-miRNA is then cleaved by the RNase Dicer into the mature approx. 22 nt long microRNA, which is incorporated into an RNA Induced Silencing Complex (RISC) with Argonaute proteins, the effector proteins of the microRNA pathway. microRNA expression and function are of specific importance since they have recently been implicated in the development of cancer and other diseases.

We have recently identified a novel mechanism of post-transcriptional regulation of microRNA expression by Argonaute (Ago) proteins as well as a novel cleavage step in microRNA processing mediated by Argonaute-2 generating a novel precursor, the ac-pre-miRNA (Cell 2007). These novel insights into the function of Argonaute proteins and specifically Ago2 (eIF2C2) have been translated into a novel method to improve RNA interference mediated by microRNAs, shRNAs or siRNAs (PNAS 2008). Recently, we also uncovered the mechanism, how Argonaute proteins regulate mature microRNA activity (RNA Biol 2011). Importantly, we also discovered that the human Argonaute-3 protein can coordinate the strand selection of the microRNA guide and passenger strand (RNA Biol 2013). Additionally, we identified novel functionally distinct splice forms of the important microRNA processing factor Drosha (Neoplasia 2012; Nucleic Acids Res 2016).

In addition to the elucidation of the microRNA processing and regulation pathways, we have also discovered a new class of microRNAs, which are not derived from the stem of the pre-miRNA hairpin, but rather from its loop - and hence we called them loop-miRs (Nucleic Acids Res 2013).

Lastly, we were interested in the function of microRNAs as well as their regulation in cancer (Genes Dev 2013, Nucleic Acids Res 2013EMBO J 2015, Nucleic Acids Res 2016).

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