RNA biochemistry

Not only the sequence, but also the 3D structure of an RNA significantly affects posttranscriptional regulation. It is known that structures in the non-coding untranslated regions (UTRs) control the stability, translation and localization of an mRNA in the cell, while structures in premRNAs modulate the splicing process.

We use evolutionary conservation to identify regulatory active structures in the UTRs of human mRNAs. One such structure we identified, is a novel binding site of the immunoregulatory RBP Roquin. Based on this discovery, we formulated a new consensus for Roquin binding sites and used it to identify novel target genes genome-wide. Interestingly, several new binding sites are AU-pure, transiently folded, stem-loops. We found that these can serve a dual function in gene repression. Depending on their folding status, they are recognized as stem-loop by Roquin and in their linear form by single-strand binding RBPs. Currently, we are evaluating Roquin’s binding preferences and dynamics towards different structured motifs and its interplay with RBPs recognizing overlapping binding sites.

Further, we established a FACS-based high-throughput screening system that allows us to interrogate thousands of putative mRNA structures simultaneously for their effect on gene expression.

Hypoxia is a major stress signal promoting pathological processes, such as cardiovascular diseases and tumor progression. Although the transcriptional response to hypoxia is well studied, we are only beginning to understand the posttranscriptional changes it induces. By performing deep sequencing analyses in reponse to hypoxia, we catalogued posttranscriptional gene regulation events in endothelial and cancer cells.

In endothelial cells, alternative splicing creates a novel, extremely efficient protein degradation domain that functions not only in human cells, but also in yeast and bacteria. In cancer cells, changes in RBP levels orchestrate hypoxia adaptation by selectively controlling the transcript abundance and alternative splicing of hypoxia response genes. We found that the RBP MBNL2 is specifically induced to promote the proliferation and migration of hypoxic cancer cells. Importantly, we have indications that MBNL2 might affect tumorigenesis also in patients.

In future, we will test the efficacy of small molecules in preventing RNA-protein interactions and thus the adaptation of cancer cells to hypoxia and establish CRISPR/Cas-based high-throughput assays for screening alternative splicing events, whether they contribute to the survival of cells under hypoxia and thus prioritize them for mechanistic analyses and potential drug development.