Long Non-coding RNA 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 (ncRNA) 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.
We have carried out microarray-based profiling of 17000 ncRNAs in three different 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 mamma 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.
As an important novel technique for studying loss-of function phenotypes in human tumors, we developed a method to knockout long non-coding RNAs in human cancer cell lines using Zinc Finger Nucleases (Gutschner, Baas & Diederichs, Genome Res 2011).
MicroRNA Biogenesis & Regulation
Our second major research endeavor focuses on the biogenesis, processing and regulation of microRNAs. microRNAs are recently discovered 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 steps (Winter et al., 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 (Diederichs & Haber, 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 (Diederichs et al., PNAS 2008). Recently, we also uncovered the mechanism, how Argonaute proteins regulate mature microRNA activity (Winter & Diederichs, RNA Biol 2011).
Currently, we are focusing our research on these areas using molecular and cellular biology, biochemistry and mammalian cell culture methods:
1) Characterization of the novel microRNA precursor, its function and relevance for microRNA maturation and RISC activation
2) Identification of protein factors that regulate mature microRNA expression on a post-transcriptional level
3) Elucidation of the role of microRNA processing factors and microRNA expression in tumorigenesis